Next Article in Journal
The N-Terminal Part of Drosophila CP190 Is a Platform for Interaction with Multiple Architectural Proteins
Previous Article in Journal
Polyphenol Extraction from Food (by) Products by Pulsed Electric Field: A Review
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 24
Issue 21
10.3390/ijms242115916
ijms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

A Protein–Protein Interaction Analysis Suggests a Wide Range of New Functions for the p21-Activated Kinase (PAK) Ste20

by
Ifeoluwapo Matthew Joshua
1,†,
Meng Lin
2,†,
Ariestia Mardjuki
1,
Alessandra Mazzola
1,3 and
Thomas Höfken
1,2,*
1
Division of Biosciences, Brunel University London, Uxbridge UB8 3PH, UK
2
Institute of Biochemistry, Kiel University, 24118 Kiel, Germany
3
Department of Biopathology and Medical and Forensic Biotechnologies, University of Palermo, 90133 Palermo, Italy
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Int. J. Mol. Sci. 2023, 24(21), 15916; https://doi.org/10.3390/ijms242115916
Submission received: 20 September 2023 / Revised: 25 October 2023 / Accepted: 30 October 2023 / Published: 2 November 2023
(This article belongs to the Section Biochemistry)
Browse Figures
Review Reports Versions Notes

Abstract

:
The p21-activated kinases (PAKs) are important signaling proteins. They contribute to a surprisingly wide range of cellular processes and play critical roles in a number of human diseases including cancer, neurological disorders and cardiac diseases. To get a better understanding of PAK functions, mechanisms and integration of various cellular activities, we screened for proteins that bind to the budding yeast PAK Ste20 as an example, using the split-ubiquitin technique. We identified 56 proteins, most of them not described previously as Ste20 interactors. The proteins fall into a small number of functional categories such as vesicle transport and translation. We analyzed the roles of Ste20 in glucose metabolism and gene expression further. Ste20 has a well-established role in the adaptation to changing environmental conditions through the stimulation of mitogen-activated protein kinase (MAPK) pathways which eventually leads to transcription factor activation. This includes filamentous growth, an adaptation to nutrient depletion. Here we show that Ste20 also induces filamentous growth through interaction with nuclear proteins such as Sac3, Ctk1 and Hmt1, key regulators of gene expression. Combining our observations and the data published by others, we suggest that Ste20 has several new and unexpected functions.

1. Introduction

p21-activated kinases (PAKs) are important signaling molecules that are highly conserved from yeast to man [1,2,3]. These serine/threonine kinases are activated by the Rho GTPases Cdc42 and Rac. The budding yeast Saccharomyces cerevisiae expresses three PAKs, Ste20, Cla4 and Skm1, all of them downstream effectors of Cdc42 [4]. Very little is known about Skm1. In contrast, numerous functions have been attributed to Cla4, ranging from septin organization to vacuolar inheritance [4].
The third member of the budding yeast PAK family, Ste20, was initially identified as an activator of three distinct mitogen-activated protein kinase (MAPK) cascades, mediating pheromone response [5,6], filamentous growth [7,8] and the hyperosmotic stress response [9,10]. In all three pathways, Ste20 phosphorylates and thereby activates Ste11, the most upstream component of these MAPK modules [11,12]. Triggering the MAPK cascades eventually results in a change of gene expression.
In the last two decades, other Ste20 functions were identified including the regulation of apoptosis [13] and the modulation of V-ATPase activity [14]. Over time, it emerged that Ste20 is involved in quite diverse and sometimes seemingly unrelated processes. Notably, this has also been observed for PAKs in other eukaryotes [1,2,3]. The wide range of Ste20 functions is also reflected in its subcellular localization. Ste20 can be found in the cytoplasm and at the plasma membrane at sites of polarized growth at the tips of buds and mating projections [15,16]. Furthermore, Ste20 can translocate to the nucleus [13,17], and it also associates with vacuolar membranes [14].
Our knowledge of Ste20 biology has increased tremendously with the identification of some downstream effectors and elucidation of molecular mechanisms. Nevertheless, large gaps in our understanding of Ste20 processes remain. It is, for example, still not entirely clear how Ste20 integrates its diverse cellular activities. Furthermore, for some processes, such as mitotic exit, a crucial role for Ste20 has been demonstrated but the underlying molecular mechanisms remain elusive [18,19,20]. The identification and characterization of more proteins that physically interact with Ste20 would improve our understanding of the functional complexity of Ste20.
Here, we report the results of a genome-wide split-ubiquitin screen for the isolation of proteins that bind to Ste20. In total, 56 proteins were identified, including only a few known Ste20 interactors. Interactions between Ste20 and glucose metabolism enzymes and nuclear proteins were further analyzed. The results suggest a wide range of unexpected functions for Ste20, in particular in many aspects of gene expression.
Due to the high conservation of PAKs, our results may also be relevant for PAKs in higher eukaryotes. In humans, PAKs are implicated in cancer, infectious diseases, diabetes, neurological disorders and cardiac diseases [21,22]. Because of these links, a better understanding of PAK biology becomes increasingly important.

2. Results

2.1. Overview of the Ste20 Split-Ubiquitin Screen

In order to improve our understanding of Ste20 functions and mechanisms, we performed a split-ubiquitin screen to identify proteins that bind to Ste20. The split-ubiquitin technique is based on the ability of the N-terminal and C-terminal domains of ubiquitin to form a quasi-native ubiquitin (Figure 1) [23,24]. If two proteins, which are attached to the N-terminal and C-terminal portion, respectively, bind to each other, the ubiquitin halves may be forced into proximity and a ubiquitin-like molecule assembles. Ubiquitin-specific proteases, present in the cytoplasm and nucleus, recognize the reconstituted ubiquitin but not its individual halves, and cleave off a reporter that is attached to the C-terminal ubiquitin portion. The technique described here employs a modified version of Ura3, an enzyme essential for uracil biosynthesis, as reporter [24]. This Ura3 variant carries an additional arginine at the extreme N-terminus (RUra3). RUra3 attached to the C-terminal half of ubiquitin is stable and functional. In contrast, RUra3 cleaved off by a ubiquitin-specific protease is rapidly degraded because arginine is a destabilizing residue in the N-end rule pathway. Therefore, the interaction between two proteins fused to the N-terminal and C-terminal halves of ubiquitin results in uracil auxotrophy. Conversely, growth on 5-fluoroorotic acid (5-FOA) indicates a protein–protein interaction because Ura3 converts 5-FOA into the toxic compound 5-fluorouracil.
The split-ubiquitin technique has a number of advantages that make it the ideal tool for the identification of Ste20 interactors. This method is suitable for monitoring interactions with membrane proteins [23,24,25] which is critical because Ste20 associates with the plasma membrane and vacuolar membranes [14,15,16]. The split-ubiquitin assay can also detect weak and transient interactions in vivo [23,25], which allows for the isolation of substrates of the kinase Ste20.
In the screen, we identified 56 proteins as putative interactors of Ste20 (Table 1). Five proteins have been shown by other groups to bind to Ste20 (Table 2). Out of these, the interaction between Bem1 and Ste20 is well established (Table 2). Like Ste20, Bem1 has a role in the three MAPK pathways’ regulating pheromone response [26,27], filamentous growth [28] and hyperosmotic stress response [28]. As a central scaffold protein, Bem1 not only binds to Ste20 but also to the related PAK Cla4 and their upstream activator Cdc42 [26,29,30,31]. Large-scale screens have also revealed that Mlc1 and Ssb2 physically interact with Ste20, and that Spb1 and Ubx7 are phosphorylated by Ste20 (Table 2). Notably, the Bem1-Ste20 and Mlc1-Ste20 interactions observed by others were also detected using the split-ubiquitin technique (Table 2). The fact that other groups independently isolated some of the proteins described here indicates that this approach robustly identifies Ste20 interactors.
We have previously characterized the interactions between Ste20 and five proteins identified in this screen (Sut1, Ncp1, Cbr1, Erg4, Vma13) (Table 2). Importantly, it has also been shown that Ste20 modulates the processes these proteins mediate, namely sterol biosynthesis (Erg4, Cbr1 and Ncp1) [32,33], sterol uptake (Sut1) [17] and V-ATPase activity (Vma13) [14]. Taken together, we present here 46 putative Ste20 interactors not described previously.
To find out whether these interactions are physiologically relevant, we further analyzed them by including published observations. First, the interactions identified in the screen presented here seem to be highly specific. We performed another split-ubiquitin screen using the same library and Rdi1 as bait (Table S1). We have chosen Rdi1 because, like Ste20, Rdi1 binds to Cdc42 [34,35]. However, Rdi1 is not a downstream effector of Cdc42. Instead, Rdi1 regulates the localization of Cdc42 by extracting it from membranes [34,36]. Rdi1, therefore, like Ste20, localizes to the cytoplasm, the plasma membrane and vacuolar membranes [34,36,37]. Fifty different proteins have been identified in the Rdi1 split-ubiquitin screen (Table S1). Only one protein (Mlc1) came up in both screens, even though Ste20 and Rdi1 both function around Cdc42 and both localize to the same compartments. This demonstrates that the interactions described here are highly specific. Using the split-ubiquitin assay, Ste20 and Rdi1 physically interact with a very different set of proteins.
The localization of the proteins identified here also suggests that the interactions are real. Importantly, all of the Ste20 interactors can be found in compartments where they could physically interact with Ste20. The huge majority of proteins localize to the cytoplasm and/or the nucleus (Table 1). Some proteins that associate with the plasma membrane, and the membranes of the endoplasmic reticulum (ER) and vacuoles, are peripheral membrane proteins on the cytoplasmic side, such as the aforementioned Bem1 and the vacuolar V-ATPase subunit Vma13 [26,38]. For other proteins, at least a portion of the protein faces the cytoplasm. For example, a topology analysis of Ale1, an enzyme involved in glycerophospholipid biosynthesis, has shown that the protein spans the ER membrane multiple times [39]. However, Ale1 also contains several cytoplasmic loops and a cytoplasmic C-terminus that is over 100 residues long. These proteins would, therefore, be accessible for Ste20.
For three proteins (Ima5, Kin82 and Pps1), subcellular localizations are not known. However, considering their functions and physical interactions with other proteins, it seems very likely that these proteins localize to the cytoplasm and/or nucleus. For example, Pps1 is involved in DNA replication [40] and physically interacts with seven proteins (Ade13, Ccr4, Dhh1, Isw1, Mpt5, Nab2 and Ser3) all of which localize to the nucleus and/or cytoplasm [41,42,43,44,45,46]. It is, therefore, reasonable to assume that Pps1 can also be found in the nucleus and/or cytoplasm.
Ste20 plays key roles in filamentation, the pheromone response and the hyperosmotic stress response [5,6,7,8,9,10]. Initially, Ste20 has been shown to activate the respective MAPK cascades through Ste11 [11,12]. More recently, it has become clear that other Ste20 functions also contribute to these processes. For example, under hyperosmotic conditions, Ste20 phosphorylates histone H4 to attenuate transcription [47] and Mrc1 phosphorylation by Ste20 prevents genomic instability caused by the collision of replication and transcription machineries [48]. Since hyperosmotic stress response, pheromone response and filamentous growth are the best characterized processes of Ste20, we searched whether any of the proteins identified in the Ste20 split-ubiquitin screen are also involved in these processes. This is the case for 30 out of the 56 proteins identified in the screen (Table 3). Based on mutant phenotypes, twenty proteins play a role in filamentation, eleven in hyperosmotic stress response, and seven in pheromone response. This suggests considerable functional overlap between Ste20 and the newly identified interactors.
We also checked published genetic interactions. With the exception of BEM1, none of the genes identified in the split-ubiquitin screen have been reported to interact with STE20. BEM1 overexpression rescues mutant phenotypes of cells lacking STE20 [49,50]. This is not surprising since the scaffold protein Bem1 binds activated Cdc42 and several of its effectors including Ste20 and Cla4. Increased Bem1 levels could lead to the activation of a parallel pathway that shares a function with Ste20.
Negative genetic interactions between CLA4 and genes identified in the STE20 split-ubiquitin screen are also interesting. The deletion of either STE20 or CLA4 does not affect the growth rate but cells lacking both genes are inviable [51]. A negative genetic interaction between CLA4 and another gene could mean that this gene shares a function with CLA4 independently of STE20 (Figure 2). Alternatively, this gene could encode a factor that acts in the same pathway as Ste20, either as upstream activator or downstream effector (Figure 2). In total, 12 out of the 56 genes identified in the STE20 split-ubiquitin screen have previously been reported to display negative genetic interactions including synthetic lethality with CLA4 (Table 4). Importantly, none of the genes identified in the STE20 split-ubiquitin screen have been reported to exhibit negative genetic interactions with STE20, demonstrating that the genetic interactions with CLA4 are highly specific. The combination of Ste20 protein–protein interactions and CLA4 genetic interactions described here supports the notion that the corresponding proteins bind to Ste20 under physiological conditions and act in the same pathway as Ste20 (Figure 2).
The proteins identified in the Ste20 split-ubiquitin screen can be grouped into a relatively small number of functional categories such as translation and protein folding, glycolysis and vesicle transport (Table 1). This suggests that the proteins identified in the screen are not a random collection. Instead, the functional categories hint at potential cellular functions of Ste20, most of them having not been described previously. Several proteins could be in more than one functional group. For example, Sgt1 acts as a linker between Hsp90 and ubiquitin ligase complexes [52,53,54]. Another example is Sut1, a nuclear protein [55] that is listed here under lipid metabolism and homeostasis [56].
Taken together, we identified here 56 proteins that bind to Ste20. Only a very small number of these interactions (Bem1, Cbr1, Erg4, Ncp1, Sut1 and Vma13) have previously been characterized, leaving 50 uncharacterized interactions. Our analysis and published data, including subcellular localizations, genetic interactions, protein–protein interactions and mutant phenotypes, suggest that these interactions are probably physiologically relevant.

2.2. Ste20 and Glucose Metabolism

Four of the proteins identified as Ste20 interactors are glycolytic enzymes (Table 1), a quite high number considering that glycolysis is a series of only ten reactions (Figure 3). We wanted to know whether other glycolytic enzymes also bind to Ste20. Since glycolysis, gluconeogenesis and the pentose phosphate pathway are intimately linked (Figure 3) [57,58], all enzymes, major and minor isoforms [59], of these pathways were tested for interaction with Ste20. Unexpectedly, all enzymes, with the exception of the two pyruvate carboxylase isoforms Pyc1 and Pyc2, bound to Ste20 (Figure 4A). Notably, these two proteins are not specific for gluconeogenesis but are also involved in other pathways such as amino acid biosynthesis and the citric acid cycle. Despite the huge number of enzymes binding to Ste20, these interactions seem to be highly specific. Ste20 does not interact with other metabolic enzymes such as β-isopropylmalate dehydrogenase (Leu2) (Figure 4A). This enzyme catalyzes a reaction in leucine biosynthesis, a pathway that is derived from pyruvate, the end product of glycolysis. Like all glucose metabolism enzymes tested here, Leu2 also localizes to the cytoplasm [60]. Thus, Ste20 interactions seem to be limited to glucose metabolism enzymes. Furthermore, none of the glucose metabolism enzymes bound to Rdi1 (Figure 4A), demonstrating again the high specificity of these interactions. Twf1, a cytoplasmic protein involved in actin filament organization [61], which was identified in the Rdi1 split-ubiquitin screen (Table S1), was included as another control. Twf1 does not interact with Ste20 but, as expected, binds to Rdi1 (Figure 4A). Bem1 and Cdc42 also served as controls. As expected, Cdc42 binds to both Ste20 and Rdi1, whereas Bem1 only forms a complex with Ste20 but not with Rdi1. These findings are all in line with published observations (Table 2) [15,34,35,62,63] and highlight the specificity of Ste20 interactions using this assay.
It is noteworthy that the split-ubiquitin technique not only detects direct protein–protein interactions but also picks up indirect interactions [64,65]. It is, therefore, not clear whether the observed interactions with Ste20 are direct or indirect. Nevertheless, because of the high number of enzymes binding to Ste20, we tried to confirm at least one of the interactions using an independent experimental approach. To this end, immunoprecipitation of Ste20 and Pgk1, a protein that was identified in the screen, was attempted. Ste20 co-precipitated with Pgk1 (Figure 4B), suggesting that the results of the split-ubiquitin assay reflect physiological interactions.
The pentose phosphate pathway plays a crucial role in the generation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) in the cytoplasm (Figure 3) [57,58]. NADPH is required for the regeneration of reduced glutathione, one of the most important antioxidants in the cell. The synthetic compound diamide oxidizes glutathione, resulting in a decrease in the cytoplasmic pool of reduced glutathione, which causes oxidative stress [66,67]. It is, therefore, not surprising that changing the activity of glucose metabolism enzymes that can alter NADPH levels has an effect on diamide sensitivity. For example, cells with reduced catalytic activity of the glycolytic enzyme triosephosphate isomerase (Tpi1) accumulate NADPH which confers diamide resistance [68,69]. Since Ste20 binds to glucose metabolism enzymes, it is tempting to speculate that Ste20 modulates the activity of these enzymes. It was, therefore, tested whether altering STE20 levels affects diamide sensitivity. STE20 overexpression resulted in an increase in diamide sensitivity (Figure 5A), whereas cells lacking STE20 exhibited diamide resistance (Figure 5B). This suggests that Ste20 negatively modulates NADPH levels possibly through the regulation of glucose metabolism enzymes.
Ijms 24 15916 g003
Figure 3. Overview of glycolysis, gluconeogenesis and the pentose phosphate pathway. In glycolysis (left, red arrows), glucose is oxidized to pyruvate. In gluconeogenesis (left, red arrows), pyruvate is converted to glucose 6-phosphate. Most of the gluconeogenic enzymes are also used in glycolysis. Gluconeogenesis-specific enzymes are Pyc1, Pyc2, Pck1 and Fbp1. Unlike some higher eukaryotes, budding yeast has no glucose 6-phosphatase. In contrast to many higher eukaryotes, the enzymes catalyzing the conversion of pyruvate to oxaloacetate (Pyc1 and Pyc2) are not mitochondrial but cytoplasmic [70]. In the oxidative branch of the pentose phosphate pathway (right, green arrows) glucose 6-phosphate is converted into ribulose 5-phosphate. This part includes two reactions that generate NADPH. In the non-oxidative branch of the pentose phosphate pathway (right, black arrows), pentoses such as ribulose 5-phosphate and the glycolytic/gluconeogenic intermediates fructose 6-phosphate and glyceraldehyde 3-phosphate are interconverted. Some reactions are catalyzed by multiple redundant isoenzymes. In contrast, Pfk1 and Pfk2 are two distinct subunits of the hetero-oligomeric phosphofructokinase [71]. For simplification, ADP/ATP and NAD+/NADH are not shown. Single-headed arrows designate irreversible reactions, and double-headed arrows indicate reversible reactions. Proteins that were identified in the Ste20 split-ubiquitin screen are shown in green, all other proteins in blue.
Figure 3. Overview of glycolysis, gluconeogenesis and the pentose phosphate pathway. In glycolysis (left, red arrows), glucose is oxidized to pyruvate. In gluconeogenesis (left, red arrows), pyruvate is converted to glucose 6-phosphate. Most of the gluconeogenic enzymes are also used in glycolysis. Gluconeogenesis-specific enzymes are Pyc1, Pyc2, Pck1 and Fbp1. Unlike some higher eukaryotes, budding yeast has no glucose 6-phosphatase. In contrast to many higher eukaryotes, the enzymes catalyzing the conversion of pyruvate to oxaloacetate (Pyc1 and Pyc2) are not mitochondrial but cytoplasmic [70]. In the oxidative branch of the pentose phosphate pathway (right, green arrows) glucose 6-phosphate is converted into ribulose 5-phosphate. This part includes two reactions that generate NADPH. In the non-oxidative branch of the pentose phosphate pathway (right, black arrows), pentoses such as ribulose 5-phosphate and the glycolytic/gluconeogenic intermediates fructose 6-phosphate and glyceraldehyde 3-phosphate are interconverted. Some reactions are catalyzed by multiple redundant isoenzymes. In contrast, Pfk1 and Pfk2 are two distinct subunits of the hetero-oligomeric phosphofructokinase [71]. For simplification, ADP/ATP and NAD+/NADH are not shown. Single-headed arrows designate irreversible reactions, and double-headed arrows indicate reversible reactions. Proteins that were identified in the Ste20 split-ubiquitin screen are shown in green, all other proteins in blue.
Ijms 24 15916 g003
Ijms 24 15916 g004
Figure 4. Ste20 binds to glucose metabolism enzymes. (A) Ste20 interacts with enzymes of glucose metabolism using the split-ubiquitin assay. Here, 104 cells of the indicated plasmid combinations were spotted onto plates lacking histidine and leucine (+uracil) to select for the plasmids, and onto plates lacking histidine, leucine and uracil (−uracil) to monitor protein–protein interactions. Ste20 and the control protein Rdi1 are both fused to the C-terminal half of ubiquitin and the reporter RUra3 (Figure 1). The other proteins are fused to the N-terminal half of ubiquitin. Leu2, Twf1, Bem1 and Cdc42 were included as controls for Ste20 and Rdi1. (B) Co-immunoprecipitation of Ste20 and Pgk1. Cells expressing STE20-3HA, and STE20-3HA with PGK1-9myc were lysed and equal amounts of protein were precipitated with anti-myc antibodies. Immunoblots were probed with antibodies raised against the myc and HA epitope.
Figure 4. Ste20 binds to glucose metabolism enzymes. (A) Ste20 interacts with enzymes of glucose metabolism using the split-ubiquitin assay. Here, 104 cells of the indicated plasmid combinations were spotted onto plates lacking histidine and leucine (+uracil) to select for the plasmids, and onto plates lacking histidine, leucine and uracil (−uracil) to monitor protein–protein interactions. Ste20 and the control protein Rdi1 are both fused to the C-terminal half of ubiquitin and the reporter RUra3 (Figure 1). The other proteins are fused to the N-terminal half of ubiquitin. Leu2, Twf1, Bem1 and Cdc42 were included as controls for Ste20 and Rdi1. (B) Co-immunoprecipitation of Ste20 and Pgk1. Cells expressing STE20-3HA, and STE20-3HA with PGK1-9myc were lysed and equal amounts of protein were precipitated with anti-myc antibodies. Immunoblots were probed with antibodies raised against the myc and HA epitope.
Ijms 24 15916 g004
Ijms 24 15916 g005
Figure 5. STE20 levels affect diamide sensitivity. (A) Overexpression of STE20 increases diamide sensitivity. Serial dilutions (1:10) of the indicated strains were spotted on selective medium containing either no diamide or diamide at a concentration that is sublethal for the wild type (2 mM). The STE20 overexpression strain carried the multicopy plasmid pRS426 with STE20 under control of its endogenous promoter. The wild type carried the empty pRS426 plasmid. (B) STE20 deletion confers diamide resistance. Serial dilutions (1:10) of the indicated strains were spotted on YPD medium containing either no diamide or diamide at a concentration that is lethal for the wild type (3 mM).
Figure 5. STE20 levels affect diamide sensitivity. (A) Overexpression of STE20 increases diamide sensitivity. Serial dilutions (1:10) of the indicated strains were spotted on selective medium containing either no diamide or diamide at a concentration that is sublethal for the wild type (2 mM). The STE20 overexpression strain carried the multicopy plasmid pRS426 with STE20 under control of its endogenous promoter. The wild type carried the empty pRS426 plasmid. (B) STE20 deletion confers diamide resistance. Serial dilutions (1:10) of the indicated strains were spotted on YPD medium containing either no diamide or diamide at a concentration that is lethal for the wild type (3 mM).
Ijms 24 15916 g005
In summary, Ste20 binds to a large number of glucose metabolism enzymes either directly or indirectly, and these interactions are probably physiologically relevant.

2.3. Interactions between Ste20 and Nuclear Proteins

Ste20 can translocate to the nucleus where it has a range of functions, including the regulation of apoptosis through histone H2B [13], the attenuation of stress gene expression via histone H4 [47] and the modulation of the transcription of sterol uptake genes through the transcriptional regulator Sut1 [17]. Using the split-ubiquitin screen, we identified another nine nuclear proteins as Ste20 interactors (Table 1). In this study, we focus on three key regulators of various steps of gene expression: Sac3, Hmt1 and Ctk1.
Sac3 is a component of the transcription export complex 2 (TREX-2). This protein complex plays a central role in the integration of the nuclear export of mature mRNA with earlier steps in the gene expression pathway including transcription and RNA processing [72]. Sac3 serves as a scaffold that binds the other TREX-2 components, Thp1, Sus1, Sem1 and Cdc31 [73,74,75,76] (Figure 6A). The binding sites of Sac3 for mRNA, the principal mRNA export factors Mex67-Mtr2, the TREX-2 components Thp1, Sem1, Sus1 and Cdc31, the nuclear pore complexes and the Mediator complex, an essential regulator of RNA polymerase II, can all be found in Sac3 residues ~1–805 (Figure 6A). In contrast, no proteins are known to interact with the C-terminal residues ~806–1301, and no function has been attributed to this region. It is noteworthy, that in our split-ubiquitin screen, the full-length Sac3 was not identified as a Ste20 interactor, but a C-terminal Sac3 fragment comprising only residues 968–1301. However, the full-length Sac3 also binds to Ste20 (Figure 6B). Since the C-terminal 334 residues of Sac3 are sufficient for interaction with Ste20, more Sac3 fragments were generated to further narrow down the Ste20-binding site (Figure 6A). Ste20 interacted with truncated Sac3 comprising C-terminal 400, 300, 200 and 100 residues (Figure 6B). However, no binding was observed for a Sac3 fragment of the last 50 residues (Figure 6B), indicating that the 100 C-terminal Sac3 residues are sufficient and necessary for the Ste20 interaction.
Ste20 lacking its nuclear localization signal (NLS) (Ste20∆NLS) is functional but it no longer translocates to the nucleus [17]. The full-length Sac3 did not bind to Ste20∆NLS (Figure 6B), suggesting that the Ste20–Sac3 interaction takes place in the nucleus. Ste20 that lacks its NLS still binds to Bem1 (Figure 6B), an interaction that occurs at the plasma membrane [26,28], demonstrating that Ste20∆NLS is suitable for monitoring protein–protein interactions outside the nucleus. All C-terminal fragments of Sac3, apart from the smallest one, also bound to Ste20∆NLS (Figure 6B). This is not unexpected because a Sac3 NLS has been predicted for residues 701–717 [81] (Figure 6A). The truncated Sac3 versions lack this putative NLS and, therefore, presumably can be found in the cytoplasm where they could interact with Ste20∆NLS. Furthermore, Ste20 bound to the other TREX-2 subunits (Thp1, Sem1, Sus1 and Cdc31) (Figure 6B). These interactions all required the Ste20 NLS (Figure 6B).
For these experiments, Cla4 was included as a control because it is a PAK related to Ste20 [51], and like Ste20, Cla4 localizes not only to the cytoplasm and plasma membrane but also the nucleus [17,82]. Cla4 bound to the control protein Bem1, confirming a well-established link [30], but it did not interact with the full-length Sac3, any of the Sac3 fragments and the other TREX-2 components (Figure 6B). This again demonstrates the high specificity of split-ubiquitin interactions.
Several large-scale screens revealed negative genetic interactions including synthetic lethality between CLA4 and SAC3 [83,84,85,86], SUS1 [84], SEM1 [83,84,85,86,87,88,89,90] and THP1 [84] deletions. These interactions were confirmed here (Figure 6C). In our hands, the deletion of either SAC3, SEM1, SUS1 or THP1 did not affect the growth rate of these strains (Figure 6C). However, in all cases, we observed synthetic lethality when combined with the deletion of CLA4 deletion (Figure 6C). In contrast, STE20 deletion has no effect on the growth of cells also lacking SAC3, SUS1, SEM1 or THP1 (Figure 6C). As mentioned above (Figure 2), such a high specificity of genetic and protein–protein interactions suggests that Sac3 and the other TREX-2 components act in the same pathway as Ste20, possibly as downstream effectors of Ste20.
The loss of SAC3 results in a strong nuclear accumulation of mRNA, demonstrating a crucial role for Sac3 in nuclear mRNA export [73,91]. Therefore, it would seem reasonable to expect numerous serious defects in cells lacking SAC3. However, under optimal lab conditions, the SAC3 deletion strain grows like the wild type or only displays slightly reduced growth (Figure 6C and Figure 7A,C), and very few other obvious defects have been reported. In this study, we found that Sac3, like Ste20, has a role in filamentous growth, a differentiation process that is triggered by nutrient depletion. Filamentous growth can be observed in both haploids and diploids, but the stimuli that lead to it, the underlying signaling pathways and the morphological responses differ slightly in these cell types [92]. In haploid cells, the lack of a fermentable carbon source such as glucose results in an invasion of the agar substratum. This form of filamentous growth is, therefore, also known as invasive growth. Diploids grown under nitrogen limitation elongate and move away from the colony, a process that is termed pseudohyphal growth. In the homozygous SAC3 deletion strain, diploid pseudohyphal growth is completely absent (Figure 7A), and cells overexpressing SAC3 display increased pseudohyphal growth (Figure 7B). In contrast, in haploid cells, SAC3 deletion or overexpression does not affect invasive growth (Figure 7C,D). These distinct phenotypes have also been observed for the ste20∆NLS mutant. Wild type STE20 is essential for both haploid invasive and diploid pseudohyphal growth [7,8] (Figure 7A,C). Cells expressing STE20 without its NLS exhibit normal haploid invasive growth (Figure 7C) as previously shown [17]. Here, we found that, unlike the STE20 wild type allele, STE20∆NLS does not complement the diploid pseudohyphal growth defect of the homozygous STE20 deletion strain (Figure 7E). An overexpression of wild type STE20 and STE20∆NLS had no effect on filamentous growth in either haploid or diploid cells (Figure 7B,D). Taken together, this suggests that Ste20 has an essential nuclear function in diploid pseudohyphal growth but not in haploid invasive growth.
Next, we examined Hmt1, the predominant arginine methyltransferase in budding yeast [93]. Through the methylation of proteins such as Hrp1, Nab2, Npl3, Snp1, Yra1 and histone H4, Hmt1 regulates a wide range of processes in gene expression. This includes transcription elongation and termination, the splicing of pre-mRNA, the nuclear export of mRNA and the formation of silent chromatin [94,95,96,97,98,99].
Hmt1 binds to Ste20, an interaction that requires the nuclear localization of Ste20 (Figure 6B). The related PAK Cla4 does not associate with Hmt1 (Figure 6B). Like the ste20∆NLS mutant and the SAC3 deletion strain, hmt1∆ cells display normal haploid invasive growth (Figure 7C), and diploid pseudohyphal growth is completely absent in hmt1∆/hmt1∆ cells (Figure 7A). Furthermore, HMT1 overexpression results in a strong increase in diploid pseudohyphal growth (Figure 7B) but has no effect in haploid cells (Figure 7D).
Finally, we examined the link between Ctk1 and Ste20. Ctk1 is the catalytic subunit of the heterotrimeric C-terminal domain (CTD) kinase I complex [100]. It phosphorylates the CTD of Rpo21 (also known as Rpb1), the largest RNA polymerase II subunit. This CTD consists of tandem heptapeptide repeats whose dynamic phosphorylation coordinates transcription with co-transcriptional events [101]. For example, CTD phosphorylation by Ctk1 plays an important role in pre-mRNA 3′ processing [102,103].
Like the other nuclear interactors of Ste20 tested here, the binding of Ste20 to Ctk1 requires the nuclear localization of Ste20, and Ctk1 does not interact with Cla4 (Figure 6B). Since CTK1 is essential in the Σ1278b background [104], which is used for filamentous growth assays, the effect of CTK1 deletion on invasive growth and pseudohyphal growth could not be tested. As for SAC3 and HMT1, the overexpression of CTK1 increased diploid pseudohyphal growth (Figure 7B) but had no effect on haploid invasive growth (Figure 7D).
As reported previously, cells lacking CTK1 grew extremely slowly (Figure 8A) [100]. The deletion of CLA4 in the ctk1∆ strain had no effect on the growth rate but, unexpectedly, STE20 deletion rescued the severe growth defect of ctk1∆ cells (Figure 8A). When a copy of wild type STE20 was brought back to the ctk1∆ ste20∆ double mutant, growth was again greatly reduced as expected, but expression of STE20∆NLS had no effect in the CTK1 STE20 double deletion strain (Figure 8A). These observations suggest that Ste20 and Ctk1 act in the same process in the nucleus, possibly the modification of RNA polymerase II CTD. Our observations can be explained by a model in which Ste20 functions as an inhibitor of an unknown protein that, like Ctk1, modifies the CTD (Figure 8B). However, compared with Ctk1, CTD modification by this hypothetical protein is less important. Since Ctk1 plays such a crucial role in CTD phosphorylation, cells lacking CTK1 display a severe growth defect but are still viable due to activity of other kinases including the hypothetical protein. Due to the lack of Ste20 inhibition in a STE20 deletion strain, the hypothetical protein would be more active but this would not affect cell growth. However, in the ctk1∆ ste20∆ double mutant, increased activity of the unknown protein could compensate for the absence of CTK1. This model would suggest that STE20 overexpression could inhibit the unknown protein even more (Figure 8B). In the presence of CTK1, reduced activity of the hypothetical protein probably would not affect the growth rate. However, in a ctk1∆ strain, STE20 overexpression could exacerbate the growth defect of ctk1∆. To test whether this is the case, we overexpressed STE20 and STE20∆NLS using the strong, inducible GAL1 promoter. Increased STE20 levels did not affect the growth of wild type cells (Figure 8C). In the ctk1∆ strain, STE20 overexpression reduced the growth even further. Notably, the overexpression of STE20 lacking its NLS had no effect (Figure 8C). These observations are in line with the proposed model, suggesting that Ste20 has a nuclear function acting in parallel with Ctk1.
In summary, Ste20 can translocate to the nucleus where it binds with high specificity to Sac3, Hmt1 and Ctk1. These proteins are key regulators integrating various aspects of gene expression from transcription to nuclear mRNA export. Nuclear Ste20, Sac3, Hmt1 and Ctk1 all play important roles in diploid pseudohyphal growth. This functional overlap and physical, as well as genetic, interactions suggest that nuclear Ste20 acts in the same processes as Sac3, Hmt1 and Ctk1.

2.4. Other Interactions

The ribosomal protein Rpl13B and seven other proteins involved in translation and protein folding were identified in the split-ubiquitin screen as Ste20 interactors (Table 1). This was further analyzed to rule out that these interactions are just artefacts that occur when Ste20 is synthesized at ribosomes. Rpl13B, a protein of the large ribosomal subunit, and the ribosome-associated chaperones Ssb2 and Zuo1 all bound specifically to Ste20 but not to the control protein Rdi1 (Figure 9A). Rpl13A, the highly similar paralog of Rpl13B, which was included here as another control, did not interact with either Ste20 or Rdi1.
CLA4 deletion has been reported to be synthetically lethal with the deletion of seven genes encoding ribosomal subunits (RPL17A, RPL19A, RPL23A, RPL24A, RPL35A, RPS21A, RPS21B) [83,87,88]. In contrast, no genetic interactions between STE20 and any genes encoding ribosomal proteins have been published. Because of the high number of known genetic interactions between CLA4 and ribosomal genes, we also analyzed RPL13B and its paralog RPL13A. The deletion of RPL13B results in a severe growth defect, whereas rpl13a∆ cells grow like the wild type (Figure 9B). STE20 deletion in these strains does not alter the growth rate. In contrast, the rpl13b∆ cla4∆ double deletion strain is not viable (Figure 9B). This synthetic lethal interaction is highly specific because rpl13a∆ cla4∆ cells are indistinguishable from the wild type.
Another group of Ste20 interactors identified in the split-ubiquitin screen comprises five proteins with ubiquitin-related functions (Table 1). Since the screen utilizes ubiquitin and ubiquitin-specific proteases (Figure 1), it was important to find out whether the identified proteins were simply artefacts. Ubp3, a ubiquitin-specific protease [105], and Ubx7, a ubiquitin regulatory X (UBX) domain-containing protein involved in ubiquitin-dependent protein degradation [106,107], both bound to Ste20 only and not to the control protein Rdi1. This suggests that these interactions are indeed specific and independent of the experimental approach employing ubiquitin (Figure 9A).
Taken together, the high specificity of protein–protein and genetic interactions argues against the notion that the interactors identified in the Ste20 split-ubiquitin screen merely represent artefacts. Rather, the interactions seem physiological with a potential role for Ste20 modulating translation and protein folding as well as ubiquitin-dependent protein degradation.

3. Discussion

Since PAKs were first described around 30 years ago [108], considerable progress has been made in the understanding of these key signaling proteins [1,2,3,4,21,22]. However, a lot of questions still remain unanswered. In order to get a comprehensive insight into the functions and the integration of various cellular activities of a PAK, we screened for the binding partners of the budding yeast PAK Ste20 as an example. Using the split-ubiquitin technique, we identified 56 proteins that physically interact with Ste20 in this study (Table 1). Most of them have not been reported to bind to Ste20 before. This raises the question whether these are physiologically relevant interactions. We believe this is the case for several reasons. (1) Some of the interactors have independently been identified by others (Table 2). (2) Interactions between Ste20 and five proteins (Cbr1, Erg4, Ncp1, Sut1 and Vma13) described in this screen have previously been characterized by us [14,17,32,33]. (3) The split-ubiquitin interactions described here are highly specific. Another split-ubiquitin screen using the same library and Rdi1 as bait identified 50 proteins (Table S1). Out of these, only Mlc1 was also found in both screens. Other interactions presented in this study also demonstrate the high specificity of the split-ubiquitin technique. For example, Ste20 only binds to the ribosomal protein Rpl13B but not to the highly similar Rpl13A (Figure 9A). (4) All of the interactors listed here, can be found in subcellular locations where they would be able to physically interact with Ste20. (5) Ste20 has a well characterized role in the activation of MAPK pathways mediating hyperosmotic stress response, pheromone response and filamentous growth [5,6,7,8,9,10]. Previously reported mutant phenotypes indicate a role in these processes for 30 out of the 56 proteins identified here as Ste20 interactors (Table 3). In addition, we show in this study that Sac3, Hmt1 and Ctk1 also have important roles in filamentous growth. This considerable functional overlap is consistent with physical interactions between Ste20 and these proteins. (6) The simultaneous deletion of STE20 and CLA4 is lethal [51], suggesting that Ste20 has one or more functions that are essential in cells lacking CLA4. Likewise, the deletion of genes-encoding proteins that act in the same pathway as Ste20 and share a function with Cla4 can be lethal in a cla4Δ background [87]. For 12 genes identified in the Ste20 split-ubiquitin screen, negative genetic interactions with a CLA4 deletion have previously been published (Table 4). We also demonstrated here the synthetic lethality for the rpl13b∆ cla4∆ strain but not for the related rpl13a∆ cla4∆ double mutant (Figure 9B). (7) The proteins identified in the screen fall into a relatively small number of functional groups such as vesicle transport and glycolysis (Table 1), suggesting that these proteins are not random hits and that Ste20 might be involved in these processes.
According to the Saccharomyces Genome Database, 155 proteins have been described by others to physically interact with Ste20 (Table 5). Most of these interactions have been found through high-throughput screens and have not been further characterized. In some cases, the Ste20 interactors described in this study and proteins identified by others are quite similar. For example, we have found the ubiquitin-specific protease Ubp2 and the UBX domain-containing Ubx7, while others have identified two more ubiquitin-specific proteases (Ubp7 and Ubp9) and another UBX domain-containing protein (Ubx5) [109].
Like the Ste20 interactors identified by us, the huge majority of the proteins that physically interact with Ste20 described by others can also easily be grouped together according to their functions (Table 5). The functional categories for the Ste20 interactors described by us and by other groups show considerable overlap (Table 1 and Table 5). Some of these functions include well established roles for Ste20 such as MAPK signaling and cell polarity [4,110]. However, other functions, such as ribosome biogenesis and tRNA synthesis and modification, are rather unexpected and these new links are certainly worth examination in the future. Notably, some of the functional categories are connected and/or overlap. For example, vesicle transport plays an important role in cell polarity [110]. Another observation worth noting is that around half the Ste20 interactors published by others are involved in various aspects of gene expression (Table 5). The potential roles for Ste20 in the regulation of gene expression will be discussed below. To summarize, the large number of Ste20 interactors identified by us and many other groups falling into a quite small number of functional categories suggests the possible involvement of Ste20 in these processes.
We examined the link between Ste20 and glucose metabolism in a bit more detail. Here, we show that Ste20 interacts with most glucose metabolism enzymes tested (Figure 4A). Importantly, the observed protein–protein interactions are not necessarily direct as the split-ubiquitin assay picks up both direct and indirect interactions [64,65]. Even for a kinase with a large number of substrates, an association with all glucose metabolism enzymes seems unlikely. In budding yeast, glycolytic enzymes form a multi-enzyme complex which binds to actin filaments [111]. Similar glycolytic metabolons have also been described for mammalian cells [112,113]. Ste20 could, therefore, interact with only a few of the enzymes directly. The other proteins would just be part of the same protein complex. The confirmation of one of the interactions (Ste20-Pgk1) by co-immunoprecipitation (Figure 4B), the fact that Ste20 does not bind to an unrelated metabolic enzyme (Leu2) from a pathway that is linked to glucose metabolism and that is also present in the cytoplasm [60] (Figure 4A) and the fact that the control protein Rdi1 does not bind to any of the enzymes (Figure 4A) all suggest that the interactions observed here are indeed specific and occur under physiological conditions.
It is conceivable that Ste20 not only binds to these enzymes but that it could modulate glucose metabolism. Diamide resistance in the STE20 deletion strain and increased diamide sensitivity in cells overexpressing STE20 (Figure 5A,B) suggest that Ste20 has a negative impact on NADPH generation probably through the modulation of the pentose phosphate pathway. The regulation of glucose metabolism by PAKs has been described before. PAK1, the closest mammalian homologue of Ste20, interacts with two glucose metabolism enzymes, phosphoglucomutase and phosphoglycerate mutase [114,115,116]. Phosphoglucomutase catalyzes the interconversion of glucose 6-phosphate and glucose 1-phosphate, a reaction that links several pathways, for example, glycolysis/gluconeogenesis and the pentose phosphate pathway to glycogen synthesis and breakdown. The phosphorylation of phosphoglucomutase by PAK1 increases the activity of this enzyme [115]. PAK1 also phosphorylates phosphoglycerate mutase, which catalyzes the interconversion of 3-phosphoglycerate and 2-phosphoglycerate [114,116]. This phosphorylation leads to the ubiquitin-mediated degradation of the enzyme.
Interestingly, PAK1 not only modulates glucose metabolism, but it also plays a central role in other aspects of glucose homeostasis [117]. To name a few examples, PAK1 is involved in insulin secretion in pancreatic β-cells, insulin-dependent glucose uptake in skeletal muscle cells and in the production of the incretin hormone glucagon-like peptide-1 (GLP-1) in intestinal L cells [117]. These examples clearly illustrate that PAKs not only act on a few substrates but that these kinases can regulate complex biological processes such as glucose homeostasis at many different levels.
Sac3, Ctk1 and Hmt1 all play important roles in diploid pseudohyphal growth but not in haploid invasive growth (Figure 7A–D). Ste20 is essential for both pathways, which is mediated via MAPK activation at the plasma membrane [7,8,11]. We now show that the nuclear translocation of Ste20 is also required, but only for diploid pseudohyphal growth (Figure 7C,E) [17]. This suggests that Ste20, in addition to its MAPK activation, also has one or more nuclear functions specifically relevant for diploid pseudohyphal growth. Since Ste20 physically interacts with Sac3, Ctk1 and Hmt1, and because of similar phenotypes, this raises the interesting possibility that during diploid pseudohyphal growth, Ste20 could modulate the activity of Sac3, Ctk1 and Hmt1. Pseudohyphal growth is a stress response to nutrient depletion. To cope with extreme stress, cells usually instantly synthesize proteins that mediate a stress response. This is achieved by preferentially transcribing, processing, exporting and translating mRNAs that encode the proteins needed for adaptation to stress [118]. It would, therefore, make sense that in response to nutrient depletion, Ste20 modulated the activity of Sac3, Hmt1 and Ctk1 not constitutively for all mRNAs but only for the subset of transcripts that are required for pseudohyphal growth. Selective mRNA export has been reported for germinal center-associated nuclear protein (GANP), the human homologue of Sac3 [119,120]. GANP exports a subset of mRNA involved in stress response from the nucleus at a higher rate. This presumably facilitates a rapid adaptation to a change in environmental conditions. Transcript specificity has also been described for Hmt1 [121]. The mitotic exit kinase Dbf2 phosphorylates and thereby activates Hmt1 which results in the transcript-specific stabilization of cyclin CLB2 mRNA. This leads to an accumulation of Clb2 protein which then promotes entry into mitosis.
What does all this mean? A wide range of functions has been attributed to Ste20; many of them do not seem to have much in common. By combining the results presented here and data published by others, we believe that most of the Ste20 functions are not unrelated but actually complement each other. For example, a major biological function that involves Ste20 is the response to extracellular signals such as pheromones, high solute concentrations and low nutrient levels. These signals trigger the three distinct MAPK pathways mentioned above. The stimulation of these pathways by Ste20 at the plasma membrane eventually leads to transcription factor activation. To ensure an efficient gene expression, Ste20 also has a range of other functions that are independent of MAPK stimulation. This includes Ste20 phosphorylation of Mrc1, a regulatory component of the DNA replication complex [48]. This leads to the inhibition of DNA replication and avoids collision of transcription and replication machineries. This is a critical process because these collisions are a major source of genomic instability. In response to hyperosmotic stress, Ste20 also phosphorylates histone H4 to attenuate the transcription of stress genes [47]. This ensures an optimal duration of the stress response which is crucial for cell survival. As mentioned above, we suggest that Ste20 regulates efficient and selective transcription, pre-mRNA processing and nuclear mRNA export trough Ctk1, Hmt1 and Sac3. In response to nutrient deprivation and other stress, Ste20 also modulates selective mRNA degradation via the phosphorylation of Dcp2, the catalytic subunit of the mRNA decapping enzyme complex [122]. Finally, due to the large number of proteins involved in translation that physically interact with Ste20 (Table 5), it seems likely that Ste20 also plays a role in protein biosynthesis, possibly modulating translational activity. Again, this could be a transcript-specific process.
The association of Ste20 with mRNA and mRNA-binding proteins is well documented. Ste20 binds to mRNA when nutrients are limited [123], which could be relevant for inducing filamentous growth. In addition, at least 15 mRNA-binding proteins interact with Ste20 (Table 5). Some of these mRNA-binding proteins (Gsp1, Rim4, She3, Tho1) are phosphorylated by Ste20 [109], and other proteins (Ded1, Pat1, Rie1, Sbp1) are phosphorylated in a Ste20-dependent manner during pseudohyphal growth [124]. Furthermore, several mRNA-binding proteins are required for pseudohyphal growth (Ccr4, Dhh1, Lsm1, Pat1, Pbp1, Sbp1) [124]. These observations further support the idea that Ste20 could play a key role in the regulation of gene expression, in particular during pseudohyphal growth.
It is quite remarkable that Ste20 could act at so many levels of gene expression. However, this is not really surprising since this would ensure an efficient adaptation to changing environmental conditions which is crucial for cell survival. In conclusion, based on our results and published data, we suggest that Ste20 might have a range of new and hitherto unappreciated functions, in particular in all aspects of gene expression.
The results obtained in budding yeast could also be relevant for higher eukaryotes. Some of the Ste20 interactors identified here have already been shown to bind to mammalian PAK1. As mentioned above, PAK1 phosphorylates phosphoglycerate mutase [114,116]. Another example is the myosin light chain Mlc1. In higher eukaryotes, MLC is phosphorylated by PAK1 [125,126,127].

4. Materials and Methods

4.1. Yeast Strains, Plasmids and Growth Conditions

All yeast strains used in this study are listed in Table 6. The strains were in the YPH499 background, with the exception of strains used for haploid invasive growth and diploid pseudohyphal growth which were in the Σ1278b background. Yeast strains were constructed using PCR-amplified cassettes [128,129,130]. The strains were grown in standard rich medium containing 1% yeast extract, 2% peptone, 2% dextrose (YPD) or minimal synthetic complete (SC) medium. For induction of the GAL1 promoter, yeast cells were grown in medium with 2% galactose and 3% raffinose instead of glucose. Media for the split-ubiquitin screen and loss of centromeric URA3-containing plasmids were supplemented with 1 mg/mL 5-FOA and 50 µg/mL uracil. All constructs used in this work are listed in Table 7.

4.2. Split-Ubiquitin Interaction Analysis

For the split-ubiquitin screens, ste20∆ cells carrying PMET25-STE20-CUBI-RURA3 and rdi1∆ cells carrying PMET25-RDI1-CUBI-RURA3 were transformed with a split-ubiquitin library (a gift from Nils Johnsson, Ulm University, Germany) and grown on 5-FOA plates for 3 days at 30 °C. Genes were subcloned after PCR amplification to confirm the interaction. Plates lacked cysteine and methionine for expression of STE20 and RDI1 fusions that were under the control of the MET25 promoter. Plates also lacked histidine and leucine to select for the presence of fusion constructs.

4.3. Immunoprecipitation and Immunoblotting

Next, 100 mL cells in the exponential phase were grown until they reached an optical density of 1 (measured at 600 nm). Cells were harvested and washed with water. After washing with lysis buffer (20 mM Tris, pH 7.5, 100 mM sodium chloride, 10 mM EDTA, 1 mM EGTA, 5% glycerol, 1% Triton X-100), cells were disrupted with glass beads in lysis buffer and clarified using centrifugation at 13,000 rpm for 5 min. Protein concentration was determined using Bradford protein assay solution. Pgk1-9myc was immunoprecipitated by adding mouse monoclonal anti-myc antibody (9E10; Santa Cruz Biotechnology) and protein G-Sepharose (GE Healthcare). Resin was washed three times with lysis buffer, resuspended in 2× SDS sample buffer, and analyzed using immunoblotting. Mouse monoclonal anti-HA (12CA5) was obtained from Santa Cruz Biotechnology, and secondary antibodies were from Jackson ImmunoResearch Laboratories.

4.4. Filamentation Assays

For haploid invasive growth assays, 105 cells of an overnight culture were spotted on YPD or selective medium, and grown at 30 °C. Plates were photographed before and after being rinsed under a stream of deionized water. For pseudohyphal growth assays, cells were grown over night, and 100 cells were spread on solid synthetic low ammonium dextrose (SLAD) medium containing 0.67% yeast nitrogen base without amino acids and without ammonium, 2% glucose and 50 μM ammonium sulfate. Plates were incubated at 30 °C. Colonies were examined with a Zeiss Axioplan 2 microscope equipped with a 5× objective and images were captured using a ProgRes C12 Plus camera (Jenoptik, Jena, Germany).

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms242115916/s1.

Author Contributions

Conceptualization, I.M.J., M.L. and T.H.; methodology, I.M.J., M.L. and T.H.; validation, I.M.J., M.L., A.M. (Ariestia Mardjuki), A.M. (Alessandra Mazzola) and T.H.; formal analysis, I.M.J., M.L. and T.H.; investigation, I.M.J., M.L., A.M. (Ariestia Mardjuki), A.M. (Alessandra Mazzola) and T.H.; data curation, T.H.; writing—original draft preparation, T.H.; writing—review and editing, T.H.; visualization, T.H.; funding acquisition, T.H. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Deutsche Forschungsgemeinschaft (DFG), grant number HO 2098/5. The APC was funded by the Brunel Open Access Publishing Fund.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data underlying the results are available as part of the article.

Acknowledgments

We thank Elmar Schiebel (Heidelberg University, Heidelberg, Germany) for suggesting the Ste20 split-ubiquitin screen, Christopher Tiedje (Kiel University, Kiel, Germany) for sharing results of the Rdi1 split-ubiquitin screen and Nils Johnsson (Ulm University, Ulm, Germany) for providing us with the split-ubiquitin library.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Kumar, A.; Molli, P.R.; Pakala, S.B.; Bui Nguyen, T.M.; Rayala, S.K.; Kumar, R. PAK thread from amoeba to mammals. J. Cell. Biochem. 2009, 107, 579–585. [Google Scholar] [CrossRef] [PubMed]
  2. Rane, C.K.; Minden, A. P21 activated kinases: Structure, regulation, and functions. Small GTPases 2014, 5, e28003. [Google Scholar] [CrossRef] [PubMed]
  3. Kumar, R.; Sanawar, R.; Li, X.; Li, F. Structure, biochemistry, and biology of PAK kinases. Gene 2017, 605, 20–31. [Google Scholar] [CrossRef] [PubMed]
  4. Boyce, K.J.; Andrianopoulos, A. Ste20-related kinases: Effectors of signaling and morphogenesis in fungi. Trends Microbiol. 2011, 19, 400–410. [Google Scholar] [CrossRef] [PubMed]
  5. Leberer, E.; Dignard, D.; Harcus, D.; Thomas, D.Y.; Whiteway, M. The protein kinase homologue Ste20p is required to link the yeast pheromone response G-protein beta gamma subunits to downstream signalling components. EMBO J. 1992, 11, 4815–4824. [Google Scholar] [CrossRef]
  6. Ramer, S.W.; Davis, R.W. A dominant truncation allele identifies a gene, STE20, that encodes a putative protein kinase necessary for mating in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 1993, 90, 452–456. [Google Scholar] [CrossRef]
  7. Liu, H.; Styles, C.A.; Fink, G.R. Elements of the yeast pheromone response pathway required for filamentous growth of diploids. Science 1993, 262, 1741–1744. [Google Scholar] [CrossRef]
  8. Roberts, R.L.; Fink, G.R. Elements of a single MAP kinase cascade in Saccharomyces cerevisiae mediate two developmental programs in the same cell type: Mating and invasive growth. Genes Dev. 1994, 8, 2974–2985. [Google Scholar] [CrossRef]
  9. O’Rourke, S.M.; Herskowitz, I. The Hog1 MAPK prevents cross talk between the HOG and pheromone response MAPK pathways in Saccharomyces cerevisiae. Genes Dev. 1998, 12, 2874–2886. [Google Scholar] [CrossRef]
  10. Raitt, D.C.; Posas, F.; Saito, H. Yeast Cdc42 GTPase and Ste20 PAK-like kinase regulate Sho1-dependent activation of the Hog1 MAPK pathway. EMBO J. 2000, 19, 4623–4631. [Google Scholar] [CrossRef]
  11. Wu, C.; Whiteway, M.; Thomas, D.Y.; Leberer, E. Molecular characterization of Ste20p, a potential mitogen-activated protein or extracellular signal-regulated kinase kinase (MEK) kinase kinase from Saccharomyces cerevisiae. J. Biol. Chem. 1995, 270, 15984–15992. [Google Scholar] [CrossRef] [PubMed]
  12. Drogen, F.; O’Rourke, S.M.; Stucke, V.M.; Jaquenoud, M.; Neiman, A.M.; Peter, M. Phosphorylation of the MEKK Ste11p by the PAK-like kinase Ste20p is required for MAP kinase signaling in vivo. Curr. Biol. 2000, 10, 630–639. [Google Scholar] [CrossRef] [PubMed]
  13. Ahn, S.H.; Cheung, W.L.; Hsu, J.Y.; Diaz, R.L.; Smith, M.M.; Allis, C.D. Sterile 20 kinase phosphorylates histone H2B at serine 10 during hydrogen peroxide-induced apoptosis in S. cerevisiae. Cell 2005, 120, 25–36. [Google Scholar] [CrossRef] [PubMed]
  14. Lin, M.; Li, S.C.; Kane, P.M.; Höfken, T. Regulation of vacuolar H+-ATPase activity by the Cdc42 effector Ste20 in Saccharomyces cerevisiae. Eukaryot. Cell 2012, 11, 442–451. [Google Scholar] [CrossRef] [PubMed]
  15. Peter, M.; Neiman, A.M.; Park, H.O.; van Lohuizen, M.; Herskowitz, I. Functional analysis of the interaction between the small GTP binding protein Cdc42 and the Ste20 protein kinase in yeast. EMBO J. 1996, 15, 7046–7059. [Google Scholar] [CrossRef] [PubMed]
  16. Leberer, E.; Wu, C.; Leeuw, T.; Fourest-Lieuvin, A.; Segall, J.E.; Thomas, D.Y. Functional characterization of the Cdc42p binding domain of yeast Ste20p protein kinase. EMBO J. 1997, 16, 83–97. [Google Scholar] [CrossRef]
  17. Lin, M.; Unden, H.; Jacquier, N.; Schneiter, R.; Just, U.; Höfken, T. The Cdc42 effectors Ste20, Cla4, and Skm1 down-regulate the expression of genes involved in sterol uptake by a mitogen-activated protein kinase-independent pathway. Mol. Biol. Cell 2009, 20, 4826–4837. [Google Scholar] [CrossRef]
  18. Höfken, T.; Schiebel, E. A role for cell polarity proteins in mitotic exit. EMBO J. 2002, 21, 4851–4862. [Google Scholar] [CrossRef]
  19. Chiroli, E.; Fraschini, R.; Beretta, A.; Tonelli, M.; Lucchini, G.; Piatti, S. Budding yeast PAK kinases regulate mitotic exit by two different mechanisms. J. Cell Biol. 2003, 160, 857–874. [Google Scholar] [CrossRef]
  20. Caydasi, A.K.; Khmelinskii, A.; Duenas-Sanchez, R.; Kurtulmus, B.; Knop, M.; Pereira, G. Temporal and compartment-specific signals coordinate mitotic exit with spindle position. Nat. Commun. 2017, 8, 14129. [Google Scholar] [CrossRef]
  21. Rane, C.K.; Minden, A. P21 activated kinase signaling in cancer. Semin. Cancer Biol. 2019, 54, 40–49. [Google Scholar] [CrossRef] [PubMed]
  22. Liu, H.; Liu, K.; Dong, Z. The Role of p21-Activated Kinases in Cancer and Beyond: Where Are We Heading? Front. Cell Dev. Biol. 2021, 9, 641381. [Google Scholar] [CrossRef] [PubMed]
  23. Johnsson, N.; Varshavsky, A. Split ubiquitin as a sensor of protein interactions in vivo. Proc. Natl. Acad. Sci. USA 1994, 91, 10340–10344. [Google Scholar] [CrossRef]
  24. Wittke, S.; Lewke, N.; Müller, S.; Johnsson, N. Probing the molecular environment of membrane proteins in vivo. Mol. Biol. Cell 1999, 10, 2519–2530. [Google Scholar] [CrossRef] [PubMed]
  25. Wittke, S.; Dünnwald, M.; Johnsson, N. Sec62p, a component of the endoplasmic reticulum protein translocation machinery, contains multiple binding sites for the Sec-complex. Mol. Biol. Cell 2000, 11, 3859–3871. [Google Scholar] [CrossRef]
  26. Leeuw, T.; Fourest-Lieuvin, A.; Wu, C.; Chenevert, J.; Clark, K.; Whiteway, M.; Thomas, D.Y.; Leberer, E. Pheromone response in yeast: Association of Bem1p with proteins of the MAP kinase cascade and actin. Science 1995, 270, 1210–1213. [Google Scholar] [CrossRef]
  27. Lyons, D.M.; Mahanty, S.K.; Choi, K.Y.; Manandhar, M.; Elion, E.A. The SH3-domain protein Bem1 coordinates mitogen-activated protein kinase cascade activation with cell cycle control in Saccharomyces cerevisiae. Mol. Cell. Biol. 1996, 16, 4095–4106. [Google Scholar] [CrossRef]
  28. Winters, M.J.; Pryciak, P.M. Interaction with the SH3 domain protein Bem1 regulates signaling by the Saccharomyces cerevisiae p21-activated kinase Ste20. Mol. Cell. Biol. 2005, 25, 2177–2190. [Google Scholar] [CrossRef]
  29. Peterson, J.; Zheng, Y.; Bender, L.; Myers, A.; Cerione, R.; Bender, A. Interactions between the bud emergence proteins Bem1p and Bem2p and Rho-type GTPases in yeast. J. Cell Biol. 1994, 127, 1395–1406. [Google Scholar] [CrossRef]
  30. Bose, I.; Irazoqui, J.E.; Moskow, J.J.; Bardes, E.S.; Zyla, T.R.; Lew, D.J. Assembly of scaffold-mediated complexes containing Cdc42p, the exchange factor Cdc24p, and the effector Cla4p required for cell cycle-regulated phosphorylation of Cdc24p. J. Biol. Chem. 2001, 276, 7176–7186. [Google Scholar] [CrossRef]
  31. Drees, B.L.; Sundin, B.; Brazeau, E.; Caviston, J.P.; Chen, G.C.; Guo, W.; Kozminski, K.G.; Lau, M.W.; Moskow, J.J.; Tong, A.; et al. A protein interaction map for cell polarity development. J. Cell Biol. 2001, 154, 549–571. [Google Scholar] [CrossRef] [PubMed]
  32. Tiedje, C.; Holland, D.G.; Just, U.; Höfken, T. Proteins involved in sterol synthesis interact with Ste20 and regulate cell polarity. J. Cell Sci. 2007, 120, 3613–3624. [Google Scholar] [CrossRef] [PubMed]
  33. Lin, M.; Grillitsch, K.; Daum, G.; Just, U.; Höfken, T. Modulation of sterol homeostasis by the Cdc42p effectors Cla4p and Ste20p in the yeast Saccharomyces cerevisiae. FEBS J. 2009, 276, 7253–7264. [Google Scholar] [CrossRef] [PubMed]
  34. Koch, G.; Tanaka, K.; Masuda, T.; Yamochi, W.; Nonaka, H.; Takai, Y. Association of the Rho family small GTP-binding proteins with Rho GDP dissociation inhibitor (Rho GDI) in Saccharomyces cerevisiae. Oncogene 1997, 15, 417–422. [Google Scholar] [CrossRef]
  35. Uetz, P.; Giot, L.; Cagney, G.; Mansfield, T.A.; Judson, R.S.; Knight, J.R.; Lockshon, D.; Narayan, V.; Srinivasan, M.; Pochart, P.; et al. A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae. Nature 2000, 403, 623–627. [Google Scholar] [CrossRef] [PubMed]
  36. Eitzen, G.; Thorngren, N.; Wickner, W. Rho1p and Cdc42p act after Ypt7p to regulate vacuole docking. EMBO J. 2001, 20, 5650–5656. [Google Scholar] [CrossRef]
  37. Richman, T.J.; Toenjes, K.A.; Morales, S.E.; Cole, K.C.; Wasserman, B.T.; Taylor, C.M.; Koster, J.A.; Whelihan, M.F.; Johnson, D.I. Analysis of cell-cycle specific localization of the Rdi1p RhoGDI and the structural determinants required for Cdc42p membrane localization and clustering at sites of polarized growth. Curr. Genet. 2004, 45, 339–349. [Google Scholar] [CrossRef]
  38. Ho, M.N.; Hirata, R.; Umemoto, N.; Ohya, Y.; Takatsuki, A.; Stevens, T.H.; Anraku, Y. VMA13 encodes a 54-kDa vacuolar H(+)-ATPase subunit required for activity but not assembly of the enzyme complex in Saccharomyces cerevisiae. J. Biol. Chem. 1993, 268, 18286–18292. [Google Scholar] [CrossRef]
  39. Pagac, M.; de la Mora, H.V.; Duperrex, C.; Roubaty, C.; Vionnet, C.; Conzelmann, A. Topology of 1-acyl-sn-glycerol-3-phosphate acyltransferases SLC1 and ALE1 and related membrane-bound O-acyltransferases (MBOATs) of Saccharomyces cerevisiae. J. Biol. Chem. 2011, 286, 36438–36447. [Google Scholar] [CrossRef]
  40. Ernsting, B.R.; Dixon, J.E. The PPS1 gene of Saccharomyces cerevisiae codes for a dual specificity protein phosphatase with a role in the DNA synthesis phase of the cell cycle. J. Biol. Chem. 1997, 272, 9332–9343. [Google Scholar] [CrossRef]
  41. Ho, Y.; Gruhler, A.; Heilbut, A.; Bader, G.D.; Moore, L.; Adams, S.L.; Millar, A.; Taylor, P.; Bennett, K.; Boutilier, K.; et al. Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry. Nature 2002, 415, 180–183. [Google Scholar] [CrossRef] [PubMed]
  42. Yu, H.; Braun, P.; Yildirim, M.A.; Lemmens, I.; Venkatesan, K.; Sahalie, J.; Hirozane-Kishikawa, T.; Gebreab, F.; Li, N.; Simonis, N.; et al. High-quality binary protein interaction map of the yeast interactome network. Science 2008, 322, 104–110. [Google Scholar] [CrossRef] [PubMed]
  43. Batisse, J.; Batisse, C.; Budd, A.; Böttcher, B.; Hurt, E. Purification of nuclear poly(A)-binding protein Nab2 reveals association with the yeast transcriptome and a messenger ribonucleoprotein core structure. J. Biol. Chem. 2009, 284, 34911–34917. [Google Scholar] [CrossRef] [PubMed]
  44. Babour, A.; Shen, Q.; Dos-Santos, J.; Murray, S.; Gay, A.; Challal, D.; Fasken, M.; Palancade, B.; Corbett, A.; Libri, D.; et al. The Chromatin Remodeler ISW1 Is a Quality Control Factor that Surveys Nuclear mRNP Biogenesis. Cell 2016, 167, 1201–1214.e15. [Google Scholar] [CrossRef]
  45. Jungfleisch, J.; Nedialkova, D.D.; Dotu, I.; Sloan, K.E.; Martinez-Bosch, N.; Brüning, L.; Raineri, E.; Navarro, P.; Bohnsack, M.T.; Leidel, S.A.; et al. A novel translational control mechanism involving RNA structures within coding sequences. Genome Res. 2017, 27, 95–106. [Google Scholar] [CrossRef]
  46. Miller, J.E.; Zhang, L.; Jiang, H.; Li, Y.; Pugh, B.F.; Reese, J.C. Genome-Wide Mapping of Decay Factor-mRNA Interactions in Yeast Identifies Nutrient-Responsive Transcripts as Targets of the Deadenylase Ccr4. G3 2018, 8, 315–330. [Google Scholar] [CrossRef]
  47. Viéitez, C.; Martínez-Cebrián, G.; Solé, C.; Böttcher, R.; Potel, C.M.; Savitski, M.M.; Onnebo, S.; Fabregat, M.; Shilatifard, A.; Posas, F.; et al. A genetic analysis reveals novel histone residues required for transcriptional reprogramming upon stress. Nucleic Acids Res. 2020, 48, 3455–3475. [Google Scholar] [CrossRef]
  48. Duch, A.; Canal, B.; Barroso, S.I.; García-Rubio, M.; Seisenbacher, G.; Aguilera, A.; de Nadal, E.; Posas, F. Multiple signaling kinases target Mrc1 to prevent genomic instability triggered by transcription-replication conflicts. Nat. Commun. 2018, 9, 379. [Google Scholar] [CrossRef]
  49. Leberer, E.; Chenevert, J.; Leeuw, T.; Harcus, D.; Herskowitz, I.; Thomas, D.Y. Genetic interactions indicate a role for Mdg1p and the SH3 domain protein Bem1p in linking the G-protein mediated yeast pheromone signalling pathway to regulators of cell polarity. Mol. Gen. Genet. 1996, 252, 608–621. [Google Scholar] [CrossRef]
  50. Höfken, T.; Schiebel, E. Novel regulation of mitotic exit by the Cdc42 effectors Gic1 and Gic2. J. Cell Biol. 2004, 164, 219–231. [Google Scholar] [CrossRef]
  51. Cvrcková, F.; De Virgilio, C.; Manser, E.; Pringle, J.R.; Nasmyth, K. Ste20-like protein kinases are required for normal localization of cell growth and for cytokinesis in budding yeast. Genes Dev. 1995, 9, 1817–1830. [Google Scholar] [CrossRef] [PubMed]
  52. Kitagawa, K.; Skowyra, D.; Elledge, S.J.; Harper, J.W.; Hieter, P. SGT1 encodes an essential component of the yeast kinetochore assembly pathway and a novel subunit of the SCF ubiquitin ligase complex. Mol. Cell 1999, 4, 21–33. [Google Scholar] [CrossRef]
  53. Hubert, D.A.; Tornero, P.; Belkhadir, Y.; Krishna, P.; Takahashi, A.; Shirasu, K.; Dangl, J.L. Cytosolic HSP90 associates with and modulates the Arabidopsis RPM1 disease resistance protein. EMBO J. 2003, 22, 5679–5689. [Google Scholar] [CrossRef] [PubMed]
  54. Takahashi, A.; Casais, C.; Ichimura, K.; Shirasu, K. HSP90 interacts with RAR1 and SGT1 and is essential for RPS2-mediated disease resistance in Arabidopsis. Proc. Natl. Acad. Sci. USA 2003, 100, 11777–11782. [Google Scholar] [CrossRef] [PubMed]
  55. Ness, F.; Bourot, S.; Régnacq, M.; Spagnoli, R.; Bergès, T.; Karst, F. SUT1 is a putative Zn[II]2Cys6-transcription factor whose upregulation enhances both sterol uptake and synthesis in aerobically growing Saccharomyces cerevisiae cells. Eur. J. Biochem. 2001, 268, 1585–1595. [Google Scholar] [CrossRef]
  56. Bourot, S.; Karst, F. Isolation and characterization of the Saccharomyces cerevisiae SUT1 gene involved in sterol uptake. Gene 1995, 165, 97–102. [Google Scholar] [CrossRef]
  57. Stincone, A.; Prigione, A.; Cramer, T.; Wamelink, M.M.; Campbell, K.; Cheung, E.; Olin-Sandoval, V.; Grüning, N.M.; Krüger, A.; Tauqeer Alam, M.; et al. The return of metabolism: Biochemistry and physiology of the pentose phosphate pathway. Biol. Rev. Camb. Philos. Soc. 2015, 90, 927–963. [Google Scholar] [CrossRef]
  58. Bertels, L.K.; Fernández Murillo, L.; Heinisch, J.J. The Pentose Phosphate Pathway in Yeasts-More Than a Poor Cousin of Glycolysis. Biomolecules 2021, 11, 725. [Google Scholar] [CrossRef]
  59. Solis-Escalante, D.; Kuijpers, N.G.; Barrajon-Simancas, N.; van den Broek, M.; Pronk, J.T.; Daran, J.M.; Daran-Lapujade, P. A Minimal Set of Glycolytic Genes Reveals Strong Redundancies in Saccharomyces cerevisiae Central Metabolism. Eukaryot. Cell 2015, 14, 804–816. [Google Scholar] [CrossRef]
  60. Ryan, E.D.; Tracy, J.W.; Kohlhaw, G.B. Subcellular localization of the leucine biosynthetic enzymes in yeast. J. Bacteriol. 1973, 116, 222–225. [Google Scholar] [CrossRef]
  61. Goode, B.L.; Drubin, D.G.; Lappalainen, P. Regulation of the cortical actin cytoskeleton in budding yeast by twinfilin, a ubiquitous actin monomer-sequestering protein. J. Cell Biol. 1998, 142, 723–733. [Google Scholar] [CrossRef] [PubMed]
  62. Simon, M.N.; De Virgilio, C.; Souza, B.; Pringle, J.R.; Abo, A.; Reed, S.I. Role for the Rho-family GTPase Cdc42 in yeast mating-pheromone signal pathway. Nature 1995, 376, 702–705. [Google Scholar] [CrossRef] [PubMed]
  63. Zhao, Z.S.; Leung, T.; Manser, E.; Lim, L. Pheromone signalling in Saccharomyces cerevisiae requires the small GTP-binding protein Cdc42p and its activator CDC24. Mol. Cell. Biol. 1995, 15, 5246–5257. [Google Scholar] [CrossRef] [PubMed]
  64. Hruby, A.; Zapatka, M.; Heucke, S.; Rieger, L.; Wu, Y.; Nussbaumer, U.; Timmermann, S.; Dünkler, A.; Johnsson, N. A constraint network of interactions: Protein-protein interaction analysis of the yeast type II phosphatase Ptc1p and its adaptor protein Nbp2p. J. Cell Sci. 2011, 124, 35–46. [Google Scholar] [CrossRef]
  65. Johnsson, N. Analyzing protein-protein interactions in the post-interactomic era. Are we ready for the endgame? Biochem. Biophys. Res. Commun. 2014, 445, 739–745. [Google Scholar] [CrossRef]
  66. Kosower, N.S.; Kosower, E.M.; Wertheim, B.; Correa, W.S. Diamide, a new reagent for the intracellular oxidation of glutathione to the disulfide. Biochem. Biophys. Res. Commun. 1969, 37, 593–596. [Google Scholar] [CrossRef]
  67. Kosower, N.S.; Kosower, E.M. Diamide: An oxidant probe for thiols. Methods Enzymol. 1995, 251, 123–133. [Google Scholar] [CrossRef]
  68. Ralser, M.; Heeren, G.; Breitenbach, M.; Lehrach, H.; Krobitsch, S. Triose phosphate isomerase deficiency is caused by altered dimerization—Not catalytic inactivity—Of the mutant enzymes. PLoS ONE 2006, 1, e30. [Google Scholar] [CrossRef]
  69. Ralser, M.; Wamelink, M.M.; Kowald, A.; Gerisch, B.; Heeren, G.; Struys, E.A.; Klipp, E.; Jakobs, C.; Breitenbach, M.; Lehrach, H.; et al. Dynamic rerouting of the carbohydrate flux is key to counteracting oxidative stress. J. Biol. 2007, 6, 10. [Google Scholar] [CrossRef]
  70. Pronk, J.T.; Yde Steensma, H.; Van Dijken, J.P. Pyruvate metabolism in Saccharomyces cerevisiae. Yeast 1996, 12, 1607–1633. [Google Scholar] [CrossRef]
  71. Kopperschläger, G.; Bär, J.; Nissler, K.; Hofmann, E. Physicochemical parameters and subunit composition of yeast phosphofructokinase. Eur. J. Biochem. 1977, 81, 317–325. [Google Scholar] [CrossRef]
  72. Stewart, M. Structure and Function of the TREX-2 Complex. Subcell. Biochem. 2019, 93, 461–470. [Google Scholar] [CrossRef] [PubMed]
  73. Fischer, T.; Strässer, K.; Rácz, A.; Rodriguez-Navarro, S.; Oppizzi, M.; Ihrig, P.; Lechner, J.; Hurt, E. The mRNA export machinery requires the novel Sac3p-Thp1p complex to dock at the nucleoplasmic entrance of the nuclear pores. EMBO J. 2002, 21, 5843–5852. [Google Scholar] [CrossRef] [PubMed]
  74. Fischer, T.; Rodríguez-Navarro, S.; Pereira, G.; Rácz, A.; Schiebel, E.; Hurt, E. Yeast centrin Cdc31 is linked to the nuclear mRNA export machinery. Nat. Cell Biol. 2004, 6, 840–848. [Google Scholar] [CrossRef] [PubMed]
  75. Rodríguez-Navarro, S.; Fischer, T.; Luo, M.J.; Antúnez, O.; Brettschneider, S.; Lechner, J.; Pérez-Ortín, J.E.; Reed, R.; Hurt, E. Sus1, a functional component of the SAGA histone acetylase complex and the nuclear pore-associated mRNA export machinery. Cell 2004, 116, 75–86. [Google Scholar] [CrossRef]
  76. Faza, M.B.; Kemmler, S.; Jimeno, S.; González-Aguilera, C.; Aguilera, A.; Hurt, E.; Panse, V.G. Sem1 is a functional component of the nuclear pore complex-associated messenger RNA export machinery. J. Cell Biol. 2009, 184, 833–846. [Google Scholar] [CrossRef]
  77. Dimitrova, L.; Valkov, E.; Aibara, S.; Flemming, D.; McLaughlin, S.H.; Hurt, E.; Stewart, M. Structural Characterization of the Chaetomium thermophilum TREX-2 Complex and its Interaction with the mRNA Nuclear Export Factor Mex67:Mtr2. Structure 2015, 23, 1246–1257. [Google Scholar] [CrossRef]
  78. Ellisdon, A.M.; Dimitrova, L.; Hurt, E.; Stewart, M. Structural basis for the assembly and nucleic acid binding of the TREX-2 transcription-export complex. Nat. Struct. Mol. Biol. 2012, 19, 328–336. [Google Scholar] [CrossRef]
  79. Gordon, J.M.B.; Aibara, S.; Stewart, M. Structure of the Sac3 RNA-binding M-region in the Saccharomyces cerevisiae TREX-2 complex. Nucleic Acids Res. 2017, 45, 5577–5585. [Google Scholar] [CrossRef]
  80. Jani, D.; Lutz, S.; Marshall, N.J.; Fischer, T.; Köhler, A.; Ellisdon, A.M.; Hurt, E.; Stewart, M. Sus1, Cdc31, and the Sac3 CID region form a conserved interaction platform that promotes nuclear pore association and mRNA export. Mol. Cell 2009, 33, 727–737. [Google Scholar] [CrossRef]
  81. Bauer, A.; Kölling, R. Characterization of the SAC3 gene of Saccharomyces cerevisiae. Yeast 1996, 12, 965–975. [Google Scholar] [CrossRef]
  82. Holly, S.P.; Blumer, K.J. PAK-family kinases regulate cell and actin polarization throughout the cell cycle of Saccharomyces cerevisiae. J. Cell Biol. 1999, 147, 845–856. [Google Scholar] [CrossRef]
  83. Costanzo, M.; VanderSluis, B.; Koch, E.N.; Baryshnikova, A.; Pons, C.; Tan, G.; Wang, W.; Usaj, M.; Hanchard, J.; Lee, S.D.; et al. A global genetic interaction network maps a wiring diagram of cellular function. Science 2016, 353, aaf1420. [Google Scholar] [CrossRef] [PubMed]
  84. Collins, S.R.; Miller, K.M.; Maas, N.L.; Roguev, A.; Fillingham, J.; Chu, C.S.; Schuldiner, M.; Gebbia, M.; Recht, J.; Shales, M.; et al. Functional dissection of protein complexes involved in yeast chromosome biology using a genetic interaction map. Nature 2007, 446, 806–810. [Google Scholar] [CrossRef] [PubMed]
  85. Costanzo, M.; Baryshnikova, A.; Bellay, J.; Kim, Y.; Spear, E.D.; Sevier, C.S.; Ding, H.; Koh, J.L.; Toufighi, K.; Mostafavi, S.; et al. The genetic landscape of a cell. Science 2010, 327, 425–431. [Google Scholar] [CrossRef]
  86. Sharifpoor, S.; van Dyk, D.; Costanzo, M.; Baryshnikova, A.; Friesen, H.; Douglas, A.C.; Youn, J.Y.; VanderSluis, B.; Myers, C.L.; Papp, B.; et al. Functional wiring of the yeast kinome revealed by global analysis of genetic network motifs. Genome Res. 2012, 22, 791–801. [Google Scholar] [CrossRef]
  87. Goehring, A.S.; Mitchell, D.A.; Tong, A.H.; Keniry, M.E.; Boone, C.; Sprague, G.F., Jr. Synthetic lethal analysis implicates Ste20p, a p21-activated potein kinase, in polarisome activation. Mol. Biol. Cell 2003, 14, 1501–1516. [Google Scholar] [CrossRef] [PubMed]
  88. Tong, A.H.; Lesage, G.; Bader, G.D.; Ding, H.; Xu, H.; Xin, X.; Young, J.; Berriz, G.F.; Brost, R.L.; Chang, M.; et al. Global mapping of the yeast genetic interaction network. Science 2004, 303, 808–813. [Google Scholar] [CrossRef]
  89. Aguilar, P.S.; Fröhlich, F.; Rehman, M.; Shales, M.; Ulitsky, I.; Olivera-Couto, A.; Braberg, H.; Shamir, R.; Walter, P.; Mann, M.; et al. A plasma-membrane E-MAP reveals links of the eisosome with sphingolipid metabolism and endosomal trafficking. Nat. Struct. Mol. Biol. 2010, 17, 901–908. [Google Scholar] [CrossRef]
  90. Fiedler, D.; Braberg, H.; Mehta, M.; Chechik, G.; Cagney, G.; Mukherjee, P.; Silva, A.C.; Shales, M.; Collins, S.R.; van Wageningen, S.; et al. Functional organization of the S. cerevisiae phosphorylation network. Cell 2009, 136, 952–963. [Google Scholar] [CrossRef]
  91. Lei, E.P.; Stern, C.A.; Fahrenkrog, B.; Krebber, H.; Moy, T.I.; Aebi, U.; Silver, P.A. Sac3 is an mRNA export factor that localizes to cytoplasmic fibrils of nuclear pore complex. Mol. Biol. Cell 2003, 14, 836–847. [Google Scholar] [CrossRef] [PubMed]
  92. Cullen, P.J.; Sprague, G.F., Jr. The regulation of filamentous growth in yeast. Genetics 2012, 190, 23–49. [Google Scholar] [CrossRef] [PubMed]
  93. Gary, J.D.; Lin, W.J.; Yang, M.C.; Herschman, H.R.; Clarke, S. The predominant protein-arginine methyltransferase from Saccharomyces cerevisiae. J. Biol. Chem. 1996, 271, 12585–12594. [Google Scholar] [CrossRef] [PubMed]
  94. Shen, E.C.; Henry, M.F.; Weiss, V.H.; Valentini, S.R.; Silver, P.A.; Lee, M.S. Arginine methylation facilitates the nuclear export of hnRNP proteins. Genes Dev. 1998, 12, 679–691. [Google Scholar] [CrossRef] [PubMed]
  95. Green, D.M.; Marfatia, K.A.; Crafton, E.B.; Zhang, X.; Cheng, X.; Corbett, A.H. Nab2p is required for poly(A) RNA export in Saccharomyces cerevisiae and is regulated by arginine methylation via Hmt1p. J. Biol. Chem. 2002, 277, 7752–7760. [Google Scholar] [CrossRef] [PubMed]
  96. Yu, M.C.; Bachand, F.; McBride, A.E.; Komili, S.; Casolari, J.M.; Silver, P.A. Arginine methyltransferase affects interactions and recruitment of mRNA processing and export factors. Genes Dev. 2004, 18, 2024–2035. [Google Scholar] [CrossRef]
  97. Yu, M.C.; Lamming, D.W.; Eskin, J.A.; Sinclair, D.A.; Silver, P.A. The role of protein arginine methylation in the formation of silent chromatin. Genes Dev. 2006, 20, 3249–3254. [Google Scholar] [CrossRef]
  98. Chen, Y.C.; Milliman, E.J.; Goulet, I.; Côté, J.; Jackson, C.A.; Vollbracht, J.A.; Yu, M.C. Protein arginine methylation facilitates cotranscriptional recruitment of pre-mRNA splicing factors. Mol. Cell. Biol. 2010, 30, 5245–5256. [Google Scholar] [CrossRef]
  99. Wong, C.M.; Tang, H.M.; Kong, K.Y.; Wong, G.W.; Qiu, H.; Jin, D.Y.; Hinnebusch, A.G. Yeast arginine methyltransferase Hmt1p regulates transcription elongation and termination by methylating Npl3p. Nucleic Acids Res. 2010, 38, 2217–2228. [Google Scholar] [CrossRef]
  100. Lee, J.M.; Greenleaf, A.L. CTD kinase large subunit is encoded by CTK1, a gene required for normal growth of Saccharomyces cerevisiae. Gene Expr. 1991, 1, 149–167. [Google Scholar]
  101. Jeronimo, C.; Collin, P.; Robert, F. The RNA Polymerase II CTD: The Increasing Complexity of a Low-Complexity Protein Domain. J. Mol. Biol. 2016, 428, 2607–2622. [Google Scholar] [CrossRef] [PubMed]
  102. Skaar, D.A.; Greenleaf, A.L. The RNA polymerase II CTD kinase CTDK-I affects pre-mRNA 3’ cleavage/polyadenylation through the processing component Pti1p. Mol. Cell 2002, 10, 1429–1439. [Google Scholar] [CrossRef] [PubMed]
  103. Ahn, S.H.; Kim, M.; Buratowski, S. Phosphorylation of serine 2 within the RNA polymerase II C-terminal domain couples transcription and 3’ end processing. Mol. Cell 2004, 13, 67–76. [Google Scholar] [CrossRef] [PubMed]
  104. Dowell, R.D.; Ryan, O.; Jansen, A.; Cheung, D.; Agarwala, S.; Danford, T.; Bernstein, D.A.; Rolfe, P.A.; Heisler, L.E.; Chin, B.; et al. Genotype to phenotype: A complex problem. Science 2010, 328, 469. [Google Scholar] [CrossRef]
  105. Baker, R.T.; Tobias, J.W.; Varshavsky, A. Ubiquitin-specific proteases of Saccharomyces cerevisiae. Cloning of UBP2 and UBP3, and functional analysis of the UBP gene family. J. Biol. Chem. 1992, 267, 23364–23375. [Google Scholar] [CrossRef]
  106. Decottignies, A.; Evain, A.; Ghislain, M. Binding of Cdc48p to a ubiquitin-related UBX domain from novel yeast proteins involved in intracellular proteolysis and sporulation. Yeast 2004, 21, 127–139. [Google Scholar] [CrossRef]
  107. Schuberth, C.; Richly, H.; Rumpf, S.; Buchberger, A. Shp1 and Ubx2 are adaptors of Cdc48 involved in ubiquitin-dependent protein degradation. EMBO Rep. 2004, 5, 818–824. [Google Scholar] [CrossRef]
  108. Manser, E.; Leung, T.; Salihuddin, H.; Zhao, Z.S.; Lim, L. A brain serine/threonine protein kinase activated by Cdc42 and Rac1. Nature 1994, 367, 40–46. [Google Scholar] [CrossRef]
  109. Ptacek, J.; Devgan, G.; Michaud, G.; Zhu, H.; Zhu, X.; Fasolo, J.; Guo, H.; Jona, G.; Breitkreutz, A.; Sopko, R.; et al. Global analysis of protein phosphorylation in yeast. Nature 2005, 438, 679–684. [Google Scholar] [CrossRef]
  110. Chiou, J.G.; Balasubramanian, M.K.; Lew, D.J. Cell Polarity in Yeast. Annu. Rev. Cell Dev. Biol. 2017, 33, 77–101. [Google Scholar] [CrossRef]
  111. Araiza-Olivera, D.; Chiquete-Felix, N.; Rosas-Lemus, M.; Sampedro, J.G.; Peña, A.; Mujica, A.; Uribe-Carvajal, S. A glycolytic metabolon in Saccharomyces cerevisiae is stabilized by F-actin. FEBS J. 2013, 280, 3887–3905. [Google Scholar] [CrossRef] [PubMed]
  112. Fuller, G.G.; Kim, J.K. Compartmentalization and metabolic regulation of glycolysis. J. Cell Sci. 2021, 134, jcs258469. [Google Scholar] [CrossRef] [PubMed]
  113. Zhu, Y.; Jin, L.; Shi, R.; Li, J.; Wang, Y.; Zhang, L.; Liang, C.Z.; Narayana, V.K.; De Souza, D.P.; Thorne, R.F.; et al. The long noncoding RNA glycoLINC assembles a lower glycolytic metabolon to promote glycolysis. Mol. Cell 2022, 82, 542–554.e6. [Google Scholar] [CrossRef] [PubMed]
  114. Shalom-Barak, T.; Knaus, U.G. A p21-activated kinase-controlled metabolic switch up-regulates phagocyte NADPH oxidase. J. Biol. Chem. 2002, 277, 40659–40665. [Google Scholar] [CrossRef] [PubMed]
  115. Gururaj, A.; Barnes, C.J.; Vadlamudi, R.K.; Kumar, R. Regulation of phosphoglucomutase 1 phosphorylation and activity by a signaling kinase. Oncogene 2004, 23, 8118–8127. [Google Scholar] [CrossRef] [PubMed]
  116. Mikawa, T.; Maruyama, T.; Okamoto, K.; Nakagama, H.; Lleonart, M.E.; Tsusaka, T.; Hori, K.; Murakami, I.; Izumi, T.; Takaori-Kondo, A.; et al. Senescence-inducing stress promotes proteolysis of phosphoglycerate mutase via ubiquitin ligase Mdm2. J. Cell Biol. 2014, 204, 729–745. [Google Scholar] [CrossRef]
  117. Chiang, Y.T.; Jin, T. p21-Activated protein kinases and their emerging roles in glucose homeostasis. Am. J. Physiol. Endocrinol. Metab. 2014, 306, E707–E722. [Google Scholar] [CrossRef]
  118. Bond, U. Stressed out! Effects of environmental stress on mRNA metabolism. FEMS Yeast Res. 2006, 6, 160–170. [Google Scholar] [CrossRef]
  119. Okamoto, N.; Kuwahara, K.; Ohta, K.; Kitabatake, M.; Takagi, K.; Mizuta, H.; Kondo, E.; Sakaguchi, N. Germinal center-associated nuclear protein (GANP) is inv3olved in mRNA export of Shugoshin-1 required for centromere cohesion and in sister-chromatid exchange. Genes Cells 2010, 15, 471–484. [Google Scholar] [CrossRef]
  120. Wickramasinghe, V.O.; Andrews, R.; Ellis, P.; Langford, C.; Gurdon, J.B.; Stewart, M.; Venkitaraman, A.R.; Laskey, R.A. Selective nuclear export of specific classes of mRNA from mammalian nuclei is promoted by GANP. Nucleic Acids Res. 2014, 42, 5059–5971. [Google Scholar] [CrossRef]
  121. Messier, V.; Zenklusen, D.; Michnick, S.W. A nutrient-responsive pathway that determines M phase timing through control of B-cyclin mRNA stability. Cell 2013, 153, 1080–1093. [Google Scholar] [CrossRef]
  122. Yoon, J.H.; Choi, E.J.; Parker, R. Dcp2 phosphorylation by Ste20 modulates stress granule assembly and mRNA decay in Saccharomyces cerevisiae. J. Cell Biol. 2010, 189, 813–827. [Google Scholar] [CrossRef] [PubMed]
  123. Mitchell, S.F.; Jain, S.; She, M.; Parker, R. Global analysis of yeast mRNPs. Nat. Struct. Mol. Biol. 2013, 20, 127–133. [Google Scholar] [CrossRef] [PubMed]
  124. Shively, C.A.; Kweon, H.K.; Norman, K.L.; Mellacheruvu, D.; Xu, T.; Sheidy, D.T.; Dobry, C.J.; Sabath, I.; Cosky, E.E.; Tran, E.J.; et al. Large-Scale Analysis of Kinase Signaling in Yeast Pseudohyphal Development Identifies Regulation of Ribonucleoprotein Granules. PLoS Genet. 2015, 11, e1005564. [Google Scholar] [CrossRef] [PubMed]
  125. Ramos, E.; Wysolmerski, R.B.; Masaracchia, R.A. Myosin phosphorylation by human cdc42-dependent S6/H4 kinase/gammaPAK from placenta and lymphoid cells. Recept. Signal Transduct. 1997, 7, 99–110. [Google Scholar]
  126. Chew, T.L.; Masaracchia, R.A.; Goeckeler, Z.M.; Wysolmerski, R.B. Phosphorylation of non-muscle myosin II regulatory light chain by p21-activated kinase (gamma-PAK). J. Muscle Res. Cell Motil. 1998, 19, 839–854. [Google Scholar] [CrossRef]
  127. Van Eyk, J.E.; Arrell, D.K.; Foster, D.B.; Strauss, J.D.; Heinonen, T.Y.; Furmaniak-Kazmierczak, E.; Côté, G.P.; Mak, A.S. Different molecular mechanisms for Rho family GTPase-dependent, Ca2+-independent contraction of smooth muscle. J. Biol. Chem. 1998, 273, 23433–23439. [Google Scholar] [CrossRef]
  128. Wach, A.; Brachat, A.; Alberti-Segui, C.; Rebischung, C.; Philippsen, P. Heterologous HIS3 marker and GFP reporter modules for PCR-targeting in Saccharomyces cerevisiae. Yeast 1997, 13, 1065–1075. [Google Scholar] [CrossRef]
  129. Longtine, M.S.; McKenzie, A., 3rd; Demarini, D.J.; Shah, N.G.; Wach, A.; Brachat, A.; Philippsen, P.; Pringle, J.R. Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 1998, 14, 953–961. [Google Scholar] [CrossRef]
  130. Janke, C.; Magiera, M.M.; Rathfelder, N.; Taxis, C.; Reber, S.; Maekawa, H.; Moreno-Borchart, A.; Doenges, G.; Schwob, E.; Schiebel, E.; et al. A versatile toolbox for PCR-based tagging of yeast genes: New fluorescent proteins, more markers and promoter substitution cassettes. Yeast 2004, 21, 947–962. [Google Scholar] [CrossRef]
  131. Tiedje, C.; Sakwa, I.; Just, U.; Höfken, T. The Rho GDI Rdi1 regulates Rho GTPases by distinct mechanisms. Mol. Biol. Cell 2008, 19, 2885–2896. [Google Scholar] [CrossRef] [PubMed]
  132. Sikorski, R.S.; Hieter, P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 1989, 122, 19–27. [Google Scholar] [CrossRef] [PubMed]
  133. Laser, H.; Bongards, C.; Schüller, J.; Heck, S.; Johnsson, N.; Lehming, N. A new screen for protein interactions reveals that the Saccharomyces cerevisiae high mobility group proteins Nhp6A/B are involved in the regulation of the GAL1 promoter. Proc. Natl. Acad. Sci. USA 2000, 97, 13732–13737. [Google Scholar] [CrossRef] [PubMed]
  134. Christianson, T.W.; Sikorski, R.S.; Dante, M.; Shero, J.H.; Hieter, P. Multifunctional yeast high-copy-number shuttle vectors. Gene 1992, 110, 119–122. [Google Scholar] [CrossRef] [PubMed]
Ijms 24 15916 g001
Figure 1. The split-ubiquitin technique. The N-terminal and C-terminal halves of ubiquitin (N-Ubi and C-Ubi) alone do not assemble. If the glycolytic enzyme Tdh3, shown here as an example, linked to N-Ubi binds to Ste20, which is fused to C-Ubi, both ubiquitin halves can form a quasi-native ubiquitin. This is recognized by a ubiquitin-specific protease (USP) which cleaves off the reporter RUra3 that is fused to C-Ubi-Ste20. The released RUra3, a modified version of the enzyme Ura3 with an arginine at the extreme N-terminus, is rapidly degraded by the N-end rule pathway. Interaction between Tdh3 and Ste20, therefore, results in uracil auxotrophy. Since Ura3 converts 5-fluoroorotic acid (5-FOA) into the toxic 5-fluorouracil, the binding of Tdh3 to Ste20 also results in 5-FOA resistance.
Figure 1. The split-ubiquitin technique. The N-terminal and C-terminal halves of ubiquitin (N-Ubi and C-Ubi) alone do not assemble. If the glycolytic enzyme Tdh3, shown here as an example, linked to N-Ubi binds to Ste20, which is fused to C-Ubi, both ubiquitin halves can form a quasi-native ubiquitin. This is recognized by a ubiquitin-specific protease (USP) which cleaves off the reporter RUra3 that is fused to C-Ubi-Ste20. The released RUra3, a modified version of the enzyme Ura3 with an arginine at the extreme N-terminus, is rapidly degraded by the N-end rule pathway. Interaction between Tdh3 and Ste20, therefore, results in uracil auxotrophy. Since Ura3 converts 5-fluoroorotic acid (5-FOA) into the toxic 5-fluorouracil, the binding of Tdh3 to Ste20 also results in 5-FOA resistance.
Ijms 24 15916 g001
Ijms 24 15916 g002
Figure 2. Interpretation of negative genetic interactions with CLA4. The Rho GTPase Cdc42 activates several downstream effectors, including the related p21-activated kinases (PAKs) Ste20 and Cla4. TDH3, which encodes a glycolytic enzyme, is shown here as an example for a gene that displays a negative genetic interaction with CLA4. (A) The Tdh3 protein could share a function with Cla4 and act in a pathway independently of Ste20. (B) However, since Tdh3 also physically interacts with Ste20 it is likely to act in the same pathway as Ste20, for example, as shown here, as a downstream effector.
Figure 2. Interpretation of negative genetic interactions with CLA4. The Rho GTPase Cdc42 activates several downstream effectors, including the related p21-activated kinases (PAKs) Ste20 and Cla4. TDH3, which encodes a glycolytic enzyme, is shown here as an example for a gene that displays a negative genetic interaction with CLA4. (A) The Tdh3 protein could share a function with Cla4 and act in a pathway independently of Ste20. (B) However, since Tdh3 also physically interacts with Ste20 it is likely to act in the same pathway as Ste20, for example, as shown here, as a downstream effector.
Ijms 24 15916 g002
Ijms 24 15916 g006
Figure 6. Ste20 interacts with the transcription export complex 2 (TREX-2) scaffold protein Sac3. (A) Domain organization in Sac3. The protein is 1301 residues long. N-terminal phenylalanine-glycine (FG) repeatedly binds to the heterodimeric principal mRNA export factor Mex67-Mtr2 which associates with mRNA [77]. The middle (M) region of Sac3 forms a complex with Thp1 and Sem1, creating an mRNA docking site [78,79]. The Cdc31-interacting domain (CID) region binds to Cdc31 and two copies of Sus1 [80]. Also included are a predicted bipartite nuclear localization signal (NLS) [81] and the Ste20-binding region (SBR) identified in this study. Not shown for simplification are the interactions between the CID region and nuclear pore complexes, and between the M-region and the Mediator complex, a key regulator of RNA polymerase II. TREX-2 components that bind to Sac3 are shown in green, and other proteins that interact with Sac3 are shown in blue. The lower panel presents the C-terminal Sac3 fragments used to determine the Ste20-binding site. Sac3 domain boundaries and Sac3 fragments are drawn to scale. (B) Split-ubiquitin interactions between Ste20 and nuclear proteins. Strains carrying the indicated plasmids were grown on selective medium supplemented with uracil as control, and on selective medium lacking uracil to detect protein–protein interactions. Ste20ΔNLS lacks its NLS (residues 272–288) [17]. Bem1 and Twf1 were included as positive and negative controls, respectively. (C) Genetic interactions between the PAKs STE20 and CLA4, and genes encoding TREX-2 subunits. Serial dilutions (1:10) of the indicated strains were spotted on selective medium with or without 5-FOA. Strains lacking CLA4 carried a CLA4 copy on a pRS316 plasmid. All other strains carried an empty pRS316 vector. 5-FOA selects against the URA3-based pRS316 plasmids.
Figure 6. Ste20 interacts with the transcription export complex 2 (TREX-2) scaffold protein Sac3. (A) Domain organization in Sac3. The protein is 1301 residues long. N-terminal phenylalanine-glycine (FG) repeatedly binds to the heterodimeric principal mRNA export factor Mex67-Mtr2 which associates with mRNA [77]. The middle (M) region of Sac3 forms a complex with Thp1 and Sem1, creating an mRNA docking site [78,79]. The Cdc31-interacting domain (CID) region binds to Cdc31 and two copies of Sus1 [80]. Also included are a predicted bipartite nuclear localization signal (NLS) [81] and the Ste20-binding region (SBR) identified in this study. Not shown for simplification are the interactions between the CID region and nuclear pore complexes, and between the M-region and the Mediator complex, a key regulator of RNA polymerase II. TREX-2 components that bind to Sac3 are shown in green, and other proteins that interact with Sac3 are shown in blue. The lower panel presents the C-terminal Sac3 fragments used to determine the Ste20-binding site. Sac3 domain boundaries and Sac3 fragments are drawn to scale. (B) Split-ubiquitin interactions between Ste20 and nuclear proteins. Strains carrying the indicated plasmids were grown on selective medium supplemented with uracil as control, and on selective medium lacking uracil to detect protein–protein interactions. Ste20ΔNLS lacks its NLS (residues 272–288) [17]. Bem1 and Twf1 were included as positive and negative controls, respectively. (C) Genetic interactions between the PAKs STE20 and CLA4, and genes encoding TREX-2 subunits. Serial dilutions (1:10) of the indicated strains were spotted on selective medium with or without 5-FOA. Strains lacking CLA4 carried a CLA4 copy on a pRS316 plasmid. All other strains carried an empty pRS316 vector. 5-FOA selects against the URA3-based pRS316 plasmids.
Ijms 24 15916 g006
Ijms 24 15916 g007
Figure 7. Nuclear Ste20, Sac3, Hmt1 and Ctk1 play a role in diploid pseudohyphal growth but not in haploid invasive growth. (A) SAC3 and HMT1 are essential for diploid pseudohyphal growth. The indicated strains were grown on synthetic low ammonium dextrose (SLAD) medium for 7 days. A smooth colony surface indicates the absence of pseudohyphal growth. (B) Overexpression of SAC3, HMT1 and CTK1 results in increased diploid pseudohyphal growth. All strains carried a multicopy pRS426 plasmid with the indicated gene. Cells were grown on low-nitrogen SLAD medium for 4 days. Images were taken at this early stage shortly after pseudohyphal growth started in the wild type to illustrate the stronger filamentous growth in strains overexpressing SAC3, HMT1 and CTK1. (C) Deletion of SAC3 and HMT1 does not affect haploid invasive growth. In total, 104 cells of the indicated strains were grown on selective medium. An image was taken after 5 days (normal growth). The plate was then washed in a stream of water and photographed again to reveal invasive growth. (D) Overexpression of genes encoding nuclear proteins has no effect on haploid invasive growth. Wild type cells carrying a multicopy pRS426 plasmid with the indicated genes were grown on selective medium. (E) Nuclear localization of Ste20 is required for diploid pseudohyphal growth. Cells of the indicated strains were grown on low-nitrogen SLAD medium for 7 days.
Figure 7. Nuclear Ste20, Sac3, Hmt1 and Ctk1 play a role in diploid pseudohyphal growth but not in haploid invasive growth. (A) SAC3 and HMT1 are essential for diploid pseudohyphal growth. The indicated strains were grown on synthetic low ammonium dextrose (SLAD) medium for 7 days. A smooth colony surface indicates the absence of pseudohyphal growth. (B) Overexpression of SAC3, HMT1 and CTK1 results in increased diploid pseudohyphal growth. All strains carried a multicopy pRS426 plasmid with the indicated gene. Cells were grown on low-nitrogen SLAD medium for 4 days. Images were taken at this early stage shortly after pseudohyphal growth started in the wild type to illustrate the stronger filamentous growth in strains overexpressing SAC3, HMT1 and CTK1. (C) Deletion of SAC3 and HMT1 does not affect haploid invasive growth. In total, 104 cells of the indicated strains were grown on selective medium. An image was taken after 5 days (normal growth). The plate was then washed in a stream of water and photographed again to reveal invasive growth. (D) Overexpression of genes encoding nuclear proteins has no effect on haploid invasive growth. Wild type cells carrying a multicopy pRS426 plasmid with the indicated genes were grown on selective medium. (E) Nuclear localization of Ste20 is required for diploid pseudohyphal growth. Cells of the indicated strains were grown on low-nitrogen SLAD medium for 7 days.
Ijms 24 15916 g007
Ijms 24 15916 g008
Figure 8. Genetic interactions between STE20 and CTK1. (A) STE20 deletion rescues the growth defect of cells lacking CTK1. Serial dilutions (1:10) of the indicated strains were grown on selective medium. (B) Model for genetic interactions between STE20 and CTK1. Ctk1 is a major regulator of RNA II polymerase through phosphorylation of the C-terminal domain (CTD) of the largest subunit. We propose that the CTD is also modified by a hypothetical protein X. This unknown protein is negatively regulated by Ste20. Due to the critical role that Ctk1 plays, cells lacking this kinase display a major growth defect, assuming that the hypothetical protein X is less important. Activity of this protein increases in the absence of the inhibitory effect of Ste20. This does not affect the growth rate in a ste20Δ strain. In contrast, when both STE20 and CTK1 are absent, the higher activity of the hypothetical protein can compensate for the growth defect caused by the loss of CTK1. STE20 overexpression results in reduced activity of the hypothetical protein. Again, this has no effect in the presence of Ctk1 but cells lacking CTK1 exhibit an even more severe growth defect. Line weights indicate activity levels of proteins. For simplification, Ctk2 and Ctk3, the other subunits of the CTD kinase I complex, and the RNA polymerase II subunits are not shown. The model shown here is for RNA polymerase II. As described in the discussion, this could also be RNA polymerase I. (C) Overexpression of nuclear STE20 exacerbates the growth defect of ctk1∆ cells. Serial dilutions (1:10) of the indicated strains were grown on selective medium containing glucose or galactose. The cells carried either an empty plasmid or plasmids in which STE20 or STE20ΔNLS were placed under control of the inducible GAL1 promoter.
Figure 8. Genetic interactions between STE20 and CTK1. (A) STE20 deletion rescues the growth defect of cells lacking CTK1. Serial dilutions (1:10) of the indicated strains were grown on selective medium. (B) Model for genetic interactions between STE20 and CTK1. Ctk1 is a major regulator of RNA II polymerase through phosphorylation of the C-terminal domain (CTD) of the largest subunit. We propose that the CTD is also modified by a hypothetical protein X. This unknown protein is negatively regulated by Ste20. Due to the critical role that Ctk1 plays, cells lacking this kinase display a major growth defect, assuming that the hypothetical protein X is less important. Activity of this protein increases in the absence of the inhibitory effect of Ste20. This does not affect the growth rate in a ste20Δ strain. In contrast, when both STE20 and CTK1 are absent, the higher activity of the hypothetical protein can compensate for the growth defect caused by the loss of CTK1. STE20 overexpression results in reduced activity of the hypothetical protein. Again, this has no effect in the presence of Ctk1 but cells lacking CTK1 exhibit an even more severe growth defect. Line weights indicate activity levels of proteins. For simplification, Ctk2 and Ctk3, the other subunits of the CTD kinase I complex, and the RNA polymerase II subunits are not shown. The model shown here is for RNA polymerase II. As described in the discussion, this could also be RNA polymerase I. (C) Overexpression of nuclear STE20 exacerbates the growth defect of ctk1∆ cells. Serial dilutions (1:10) of the indicated strains were grown on selective medium containing glucose or galactose. The cells carried either an empty plasmid or plasmids in which STE20 or STE20ΔNLS were placed under control of the inducible GAL1 promoter.
Ijms 24 15916 g008
Ijms 24 15916 g009
Figure 9. Ste20 binds to ribosome-associated proteins and factors involved in ubiquitin-dependent protein degradation. (A) Split-ubiquitin interactions between Ste20 and ribosome-associated proteins and proteins with ubiquitin-related functions. In this experiment, 104 cells of the indicated plasmid combinations were spotted onto control plates supplemented with uracil, and onto plates lacking uracil to monitor protein–protein interactions. Bem1 and Twf1 were included as controls for Ste20 and Rdi1. (B) Genetic interaction between CLA4 and RPL13B. Serial dilutions (1:10) of the indicated strains were spotted on selective medium with or without 5-FOA. Strains lacking CLA4 carried a CLA4 copy on a pRS316 plasmid. All other strains carried an empty pRS316 vector. 5-FOA selects for cells that have lost the URA3-based pRS316 plasmids.
Figure 9. Ste20 binds to ribosome-associated proteins and factors involved in ubiquitin-dependent protein degradation. (A) Split-ubiquitin interactions between Ste20 and ribosome-associated proteins and proteins with ubiquitin-related functions. In this experiment, 104 cells of the indicated plasmid combinations were spotted onto control plates supplemented with uracil, and onto plates lacking uracil to monitor protein–protein interactions. Bem1 and Twf1 were included as controls for Ste20 and Rdi1. (B) Genetic interaction between CLA4 and RPL13B. Serial dilutions (1:10) of the indicated strains were spotted on selective medium with or without 5-FOA. Strains lacking CLA4 carried a CLA4 copy on a pRS316 plasmid. All other strains carried an empty pRS316 vector. 5-FOA selects for cells that have lost the URA3-based pRS316 plasmids.
Ijms 24 15916 g009
Table 1. Proteins identified in the Ste20 split-ubiquitin screen. Data come from the Saccharomyces Genome Database (https://www.yeastgenome.org accessed on 1 September 2023). For simplification, subcompartments are not listed. For example, “nucleoplasm”, “nucleolus”, “kinetochore” and “CTDK-I complex” are all included in “nucleus”. A, actin cortical patch. C, cytoplasm. CW, cell wall. ER, endoplasmic reticulum. G, Golgi apparatus. LD, lipid droplet. M, mitochondrion. N, nucleus. PM, plasma membrane. R, ribosome. SPB, spindle pole body. SV, secretory vesicle. U, unknown. V, vacuole.
Table 1. Proteins identified in the Ste20 split-ubiquitin screen. Data come from the Saccharomyces Genome Database (https://www.yeastgenome.org accessed on 1 September 2023). For simplification, subcompartments are not listed. For example, “nucleoplasm”, “nucleolus”, “kinetochore” and “CTDK-I complex” are all included in “nucleus”. A, actin cortical patch. C, cytoplasm. CW, cell wall. ER, endoplasmic reticulum. G, Golgi apparatus. LD, lipid droplet. M, mitochondrion. N, nucleus. PM, plasma membrane. R, ribosome. SPB, spindle pole body. SV, secretory vesicle. U, unknown. V, vacuole.
ProteinMolecular Function and/or Biological ProcessLocalization
Glycolysis
Cdc19Pyruvate kinaseC, PM
Gpm1Phosphoglycerate mutaseC, M
Pgk1Phosphoglycerate kinaseC, M, PM
Tdh3Glyceraldehyde 3-phosphate dehydrogenaseC, CW, LD, M, N, PM
Lipid metabolism and homeostasis
Ale1Lysophospholipid acyltransferase involved in glyerophospholipid biosynthesisER
Cbr1Cytochrome b5 reductase involved in ergosterol biosynthesisN, M
Erg4C-24(28) sterol reductase involved in ergosterol biosynthesisER
Hes1Sterol transfer protein involved in exocytosis, endocytosis and maintenance of cell polarityPM, V
Ncp1NADPH-cytochrome P450 reductase involved in ergosterol biosynthesisC, ER, M
Scs7Fatty acid α-hydroxylase involved in inositol phosphoceramide metabolismER
Sut1Zn(II)2Cys6 family transcription factor that regulates sterol uptake genesC, N
Other metabolic pathways
Dfr1Dihydrofolate reductase involved in tetrahydrofolate biosynthesisC, M
Ima5Oligosaccharide α-1,6-glucosidase involved in disaccharide catabolismU
Nuclear functions
Ctk1 Catalytic subunit of C-terminal domain kinase I which phosphorylates RNA polymerase II to affect transcription and pre-mRNA 3’ end processing N
Hat2Histone H4 acetyltransferase involved in chromatin assembly, disassembly and silencingC, N
Hmt1 Protein-arginine methyltransferase involved in transcription regulation and mRNA export N
Mcm22Kinetochore protein involved in chromosome segregationN
Pta1 Subunit of mRNA cleavage and polyadenylation specificity factor N
Rpa190Subunit of RNA polymerase I involved in ribosomal RNA transcriptionN
Sac3Involved in mRNA export from the nucleusN
Sin3Component of histone deacetylase complexes involved in diverse processesN
Spb1rRNA methyltransferase involved in rRNA processing and maturation of large ribosomal subunitN
Translation and protein folding
Efb1Guanyl-nucleotide exchange factor subunit of the translation elongation factor 1 complexC
Egd2α subunit of the nascent polypeptide-associated complexC
Ncs2 Protein involved in tRNA wobble uridine thiolation and protein urmylation C
Rpl13BSubunit of the large ribosomal subunitR
Ssb2Cytoplasmic ATPase that is a ribosome-associated molecular chaperoneC, PM, R
Tif3Translation initiation factor eIF4BC
Tma22Protein of unknown function that associates with ribosomesC, R
Zuo1 Ribosome-associated chaperone C, M, N, R
Cell polarity
Abp1Protein that activates actin nucleation mediated by the Arp2/Arp3 complexA, C
Bem1Scaffold protein involved in establishing cell polarity and morphogenesisC, M, PM
Cdc15Protein kinase involved in mitotic exit, cytokinesis and meiotic spindle disassemblyC, SPB
Mlc1Myosin light chain involved in formation and contraction of actomyosin ringPM, SV
Rvs167Cytoskeletal protein involved in actin cytoskeleton reorganization and endocytosisA, C
Ubiquitin-related functions
Cdc53Subunit of Skp1/Cul1/F-box complexes required for G1/S and G2/M phase transitionsC, N
Duf1Ubiquitin-binding protein of unknown functionC
Sgt1Co-chaperone protein that associates with the Skp1/Cul1/F-box ubiquitin ligase complexC, ER
Ubp3Ubiquitin-specific protease that regulates the osmotic stress response and ER to Golgi vesicle-mediated transportC
Ubx7Protein of unknown function involved in ubiquitin-dependent protein degradationN, ER
Hsp90 functions
Aha1Co-chaperone that regulates the activity of members of the Hsp90 familyC, N
Cpr6Peptidyl-prolyl cis-trans isomerase involved in folding of several proteins including Hsp90C
Sse1 Adenyl-nucleotide exchange factor involved in protein folding and refolding C
Tah1Component of Rvb1-Rvb2-Tah1-Pih1 complex that interacts with Hsp90 to mediate assembly of large protein complexesC, N
Vesicle transport
Arf2GTPase that acts as ADP ribosylation factor involved in regulation of vesicle formation in ER to Golgi and Golgi to plasma membrane vesicular transportC, G
Get3 Guanyl-nucleotide exchange factor involved in ER protein-membrane targeting and cis-Golgi to rough ER transport C, ER
Trx2Putative disulfide oxidoreductase involved in cellular redox homeostasis and vesicle-mediated traffic between the ER and GolgiC
Vacuolar proteins
Vma13Subunit of V-ATPase involved in vacuolar acidificationV
Vtc4Polyphosphate kinase involved in vacuolar transport and vacuole fusionER, V
Transmembrane transport
Alr2Inorganic cation transmembrane transporter that moves magnesium ionsPM
Atr2Borate efflux transmembrane transporterPM
Other functions
Efr3Protein involved in protein-plasma membrane targetingM, PM
Ira1 GTPase-activating protein which regulates Ras protein signal transduction M, PM
Pps1Protein phosphatase involved in DNA replication and MAP kinase inactivationU
Kin82Putative protein kinase involved in the regulation of phospholipid translocationU
Lsb1Protein involved in regulation of Arp2/3 complex-mediated actin nucleation A, C, N,
Table 2. Published physical interactions between Ste20 and proteins identified in the split-ubiquitin screen. Data are from the Saccharomyces Genome Database (https://www.yeastgenome.org, accessed on 1 September 2023). PCA, protein-fragment complementation assay. *, split-ubiquitin assay.
Table 2. Published physical interactions between Ste20 and proteins identified in the split-ubiquitin screen. Data are from the Saccharomyces Genome Database (https://www.yeastgenome.org, accessed on 1 September 2023). PCA, protein-fragment complementation assay. *, split-ubiquitin assay.
ProteinType of Assay
Bem1Two-hybrid
Affinity capture—Western
Phosphorylation
PCA
Co-crystal structure
Reconstituted complex
PCA *
Protein-peptide
Cbr1Affinity capture—Western
Erg4Affinity capture—Western
Mlc1PCA *
Spb1Phosphorylation
Ssb2Co-purification
Sut1Affinity capture—Western
Ubx7Phosphorylation
Vma13Affinity capture—Western
Table 3. Mutant phenotypes of genes identified in the Ste20 split-ubiquitin screen. Data come from the Saccharomyces Genome Database (https://www.yeastgenome.org, accessed on 1 September 2023). With the exception of BEM1, all phenotypes are for null deletion or overexpression strains. For BEM1, several unspecified alleles (bem1-m1, bem1-m2 and bem1-s1) have also been used.
Table 3. Mutant phenotypes of genes identified in the Ste20 split-ubiquitin screen. Data come from the Saccharomyces Genome Database (https://www.yeastgenome.org, accessed on 1 September 2023). With the exception of BEM1, all phenotypes are for null deletion or overexpression strains. For BEM1, several unspecified alleles (bem1-m1, bem1-m2 and bem1-s1) have also been used.
GeneFilamentationPheromone ResponseHyperosmotic Stress Response
ABP1Null--
Overexpression
ALR2Overexpression--
ARF2Overexpression--
BEM1Nullbem1-s1Null
bem1-m1 and bem1-m2
Null
CTK1--Null
Null
DFR1Overexpression--
EFB1Null--
EFR3Overexpression--
ERG4NullNullNull
GET3-Null-
Overexpression
HES1Overexpression--
IRA1Null--
KIN82-Overexpression-
NCP1Null--
Null
Null
NCS2Overexpression--
PGK1Null--
PPS1Overexpression--
RPA190Null--
RPL13B-NullNull
RVS167--Null
Null
SAC3--Null
SCS7--Null
SIN3-Null-
SSE1--Null
SUT1OverexpressionNullNull
TIF3--Null
UBP3Overexpression--
UBX7Overexpression--
VMA13Null--
ZUO1--Null
Null
Table 4. Negative genetic interactions between CLA4 and genes identified in the STE20 split-ubiquitin screen. Data are from the Saccharomyces Genome Database (https://www.yeastgenome.org, accessed on 1 September 2023).
Table 4. Negative genetic interactions between CLA4 and genes identified in the STE20 split-ubiquitin screen. Data are from the Saccharomyces Genome Database (https://www.yeastgenome.org, accessed on 1 September 2023).
GeneType of Interaction
BEM1Synthetic lethality
Negative genetic
CDC15Negative genetic
CDC53Negative genetic
CTK1Negative genetic
ERG4Synthetic lethality
NCP1Synthetic lethality
NCS2Synthetic lethality
RVS167Synthetic growth defect
Synthetic lethality
Negative genetic
SAC3Negative genetic
SIN3Negative genetic
Synthetic lethality
SSE1Negative genetic
TDH3Negative genetic
Table 5. Biological functions of proteins interacting with Ste20. Interactors identified by others are from the Saccharomyces Genome Database (https://www.yeastgenome.org, accessed on 1 September 2023).
Table 5. Biological functions of proteins interacting with Ste20. Interactors identified by others are from the Saccharomyces Genome Database (https://www.yeastgenome.org, accessed on 1 September 2023).
Biological ProcessSte20 Interactors Identified HereSte20 Interactors Identified by Others
Lipid metabolism and homeostasisAle1, Cbr1, Erg4, Hes1, Ncp1, Scs7, Sut1Are2, Cho2, Cst26, Pah1, Sct1
Other metabolic pathwaysCdc19, Dfr1, Gpm1, Ima5, Pgk1, Tdh3Pcm1, Trp2, Yig1
Ubiquitin-related functionsCdc53, Duf1, Sgt1, Ubp3, Ubx7Aly2, Rpt5, Ubp7, Ubp9, Ubx5, Ubx7
Vesicle transport and exocytosisArf2, Get3, Trx2Apl5, Boi1, Boi2, Cog4, Ent2, Erv46, Exo84, Glo3, Msb4, Sec2, Sro7
Cell polarityAbp1, Bem1, Cdc15, Mlc1, Rvs167Aim3, Bem1, Bem4, Bni1, Bud6, Bud8, Cbk1, Cdc3, Cdc24, Cdc42, Rga2, Mlc1, Myo3
MAPK signaling-Nbp2, Ptc1, Ptp2, Sho1, Slt2, Ste11
tRNA synthesis, modification and exportPta1Ats1, Dus1, Gus1, Krs1, Ses1, Sol2
Ribosome biogenesisRpa190, Spb1Bms1, Drs1, Erb1, Nsi1, Nsr1, Rcm1, Rnh70, Rrn6, Sas10, Spb1, Utp5, Utp7
Translation and protein foldingEfb1, Egd2, Ncs2, Rpl13B, Ssb2, Sse1, Tif3, Tma22, Zuo1Asc1, Cdc33, Ebs1, Rpl25, Rpl26A, Rps1A, Rps2, Rps3, Rps6A, Rps7A, Rps8A, Rps9A, Rps10A, Rps12, Rps13, Rps14A, Rps15, Rps19A, Rps20, Rps27A, Sbp1, Ssb1, Ssb2, Ssd1
Chromatin organizationHat2, Sin3Cse4, Hhf1, Htb1, Htb2, Isw1, Rsc6, Rsc8, Spt16
TranscriptionCtk1Azf1, Bdf2, Brf1, Ccr4, Cin5, Ckb1, Crz1, Rpb5, Sgv1
Other nuclear functionsHmt1, Mcm22, Pps1Mpc54, Mps3, Mrc1, Rad1, Rad53, Spo21
mRNA bindingSac3Bfr1, Crm1, Dcp2, Dhh1, Gsp1, Hek2, Nab2, Nup53, Prp21, Puf4, Rim4, She3, Tho1, Tom1
Cell cycle-Cdc28, Clb1, Clb2, Clb5, Cln1, Cln2, Hsl7
Other functionsAha1, Alr2, Atr2, Cpr6, Efr3, Ira1, Kin82, Lsb1, Tah1, Vma13, Vtc4Atg9, Avt4, Bmh1, Bmh2, Cnm1, Ecm3, Ema35, Frt2, Hsp30, Ist2, Mdg1, Mge1, Mrpl36, Mrx9, Nvj2, Pam1, Sdd3, Stb2, Ste4, Ybr071w, Yhr131c, Yml037c, Yml083c, Yml096w
Table 6. Yeast strains used in this study.
Table 6. Yeast strains used in this study.
StrainGenotypeSource
AMY2PC344 sac3∆::hphNT1/sac3∆::KanMX6This study
CTY64YPH499 rdi1∆::KanMX6[131]
MLY208YPH499 STE20-3HA-His3MX6This study
MLY215PPY966 hmt1∆::hphNT1This study
MLY219PC344 hmt1∆::hphNT1/hmt1∆::KanMX6This study
MLY224YPH499 sac3∆::His3MX6This study
MLY225YPH499 cla4∆::klTRP1 sac3∆::His3MX6This study
MLY227YPH499 sac3∆::His3MX6 ste20∆::klTRP1This study
MLY241YPH499 ctk1∆::KanMX6 ste20∆::klTRP1This study
MLY255PPY966 sac3∆::His3MX6This study
PC344MATa/MATα ura3-52/ura3-52[131]
PPY966MATa his3::hisG leu2::hisG trp1::hisG ura3-52[32]
SHY93YPH499 ctk1∆::His3MX6This study
THY310YPH499 ste20∆::klTRP1[32]
THY608YPH499 cla4∆::klTRP1[17]
THY697PPY966 ste20∆::hphNT1[131]
THY706PC344 ste20∆::hphNT1/ste20∆::KanMX6[131]
THY878YPH499 STE20-3HA-His3MX6 PGK1-9Myc-KanMX6This study
THY879YPH499 rpl13a∆::His3MX6This study
THY880YPH499 rpl13a∆::His3MX6 ste20∆::klTRP1This study
THY883YPH499 cla4∆::klTRP1 rpl13a∆::His3MX6This study
THY885YPH499 sem1∆::His3MX6This study
THY886YPH499 sem1∆::His3MX6 ste20∆::klTRP1This study
THY887YPH499 ste20∆::klTRP1 sus1∆::His3MX6This study
THY888YPH499 sus1∆::His3MX6This study
THY889YPH499 thp1∆::KanMX6This study
THY890YPH499 ste20∆::klTRP1 thp1∆::KanMX6This study
THY891YPH499 cla4∆::klTRP1 sem1∆::His3MX6This study
THY892YPH499 cla4∆::klTRP1 sus1∆::His3MX6This study
THY894YPH499 cla4∆::klTRP1 thp1∆::KanMX6This study
THY895YPH499 rpl13b∆::His3MX6This study
THY896YPH499 ste20∆::klTRP1 rpl13b∆::His3MX6This study
THY897YPH499 cla4∆::klTRP1 rpl13b∆::His3MX6This study
YPH499MATa ura3-52 lys2-801 ade2-101 trp1∆63 his3∆200 leu2∆1[132]
Table 7. Plasmids used in this study. STE20∆NLS constructs lack base pairs 814–862 of the STE20 open reading frame encoding the NLS (residues 272–288) [6].
Table 7. Plasmids used in this study. STE20∆NLS constructs lack base pairs 814–862 of the STE20 open reading frame encoding the NLS (residues 272–288) [6].
PlasmidGenotypeSource
pADNX2 μm, LEU2, PADH1-NUbiquitin-HA[133]
pAK4pADNX carrying GPM1This study
pAK7pADNX carrying PGK1This study
pCT2pRS313 carrying PMET25-RDI1-CUbiquitin-RURA3[17]
pIJ1pADNX carrying GND2This study
pIJ2pADNX carrying FBP1This study
pIJ3pADNX carrying GLK1This study
pIJ4pADNX carrying SOL4This study
pKA86pRS316 carrying PGAL1-STE20-3HA[17]
pML70pRS313 carrying PMET25-CLA4-CUbiquitin-RURA3[33]
pML110pRS426 carrying HMT1This study
pML112pADNX carrying SAC3This study
pML118pRS313 carrying PMET25-STE20∆NLS-CUbiquitin-RURA3This study
pRS313CEN, HIS3[132]
pRS316CEN, URA3[132]
pRS4262 μm, URA3[134]
pTH102pRS316 carrying CLA4[32]
pTH197pRS313 carrying PMET25-STE20-CUbiquitin>-RURA3[32]
pTH256pRS316 carrying STE20[32]
pTH344pADNX carrying BEM1[32]
pTH351pADNX carrying CDC19This study
pTH369pADNX carrying CTK1This study
pTH371pADNX carrying HMT1This study
pTH418pADNX carrying TDH3This study
pTH424pADNX carrying SOL3This study
pTH425pADNX carrying GND1This study
pTH426pADNX carrying ZWF1This study
pTH427pADNX carrying HXK2This study
pTH428pADNX carrying TKL1This study
pTH430pADNX carrying PGI1This study
pTH431pADNX carrying PFK1This study
pTH432pADNX carrying FBA1This study
pTH434pADNX carrying ENO1This study
pTH437pADNX carrying TPI1This study
pTH438pADNX carrying TDH1This study
pTH439pADNX carrying RKI1This study
pTH440pADNX carrying RPE1This study
pTH443pADNX carrying PCK1This study
pTH444pADNX carrying PFK2This study
pTH448pADNX carrying ENO2This study
pTH449pADNX carrying TDH2This study
pTH458pADNX carrying LEU2This study
pTH459pADNX carrying TAL1This study
pTH465pADNX carrying UBP3This study
pTH466pADNX carrying TKL2This study
pTH467pADNX carrying TWF1This study
pTH469pRS426 carrying CTK1This study
pTH470pADNX carrying HXK1This study
pTH471pADNX carrying PYK2This study
pTH472pADNX carrying CDC42This study
pTH473pADNX carrying PYC1This study
pTH474pADNX carrying PYC2This study
pTH475pADNX carrying THP1This study
pTH476pADNX carrying SEM1This study
pTH477pADNX carrying SUS1This study
pTH478pADNX carrying CDC31This study
pTH479pADNX carrying RPL13AThis study
pTH480pADNX carrying RPL13BThis study
pTH482pADNX carrying ZUO1This study
pTH483pADNX carrying UBX7This study
pTH485pADNX carrying SAC3901−1301This study
pTH487pADNX carrying SSB2This study
pTH488pRS316 carrying STE20∆NLSThis study
pTH489pRS426 carrying SAC3This study
pTH490pRS426 carrying STE20This study
pTH491pRS426 carrying STE20∆NLSThis study
pTH492pRS316 carrying PGAL1-STE20∆NLS-3HAThis study
pTH494pADNX carrying SAC31001−1301This study
pTH495pADNX carrying SAC31101−1301This study
pTH496pADNX carrying SAC31201−1301This study
pTH501pADNX carrying SAC31251−1301This study
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Joshua, I.M.; Lin, M.; Mardjuki, A.; Mazzola, A.; Höfken, T. A Protein–Protein Interaction Analysis Suggests a Wide Range of New Functions for the p21-Activated Kinase (PAK) Ste20. Int. J. Mol. Sci. 2023, 24, 15916. https://doi.org/10.3390/ijms242115916

AMA Style

Joshua IM, Lin M, Mardjuki A, Mazzola A, Höfken T. A Protein–Protein Interaction Analysis Suggests a Wide Range of New Functions for the p21-Activated Kinase (PAK) Ste20. International Journal of Molecular Sciences. 2023; 24(21):15916. https://doi.org/10.3390/ijms242115916

Chicago/Turabian Style

Joshua, Ifeoluwapo Matthew, Meng Lin, Ariestia Mardjuki, Alessandra Mazzola, and Thomas Höfken. 2023. "A Protein–Protein Interaction Analysis Suggests a Wide Range of New Functions for the p21-Activated Kinase (PAK) Ste20" International Journal of Molecular Sciences 24, no. 21: 15916. https://doi.org/10.3390/ijms242115916

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop