Heterologous Expressed NbSWP12 from Microsporidia Nosema bombycis Can Bind with Phosphatidylinositol 3-Phosphate and Affect Vesicle Genesis
Abstract
:1. Introduction
2. Materials and Methods
2.1. Parasite, Cells, and Cell Culture
2.2. Multiple Sequence Alignment
2.3. Protein Expression and Purification
2.4. Non-Reducing SDS-PAGE
2.5. Yeast Two-Hybrid Analysis
2.6. Liposome Co-Sedimentation
2.7. Lipid Strip Assay
2.8. Targeting of NbSWP12 in Saccharomyces cerevisiae
2.9. Vacuole Staining
2.10. Complementation of Yeast gvp36Δ by NbSWP12
3. Results
3.1. SWP12 and S. cerevisiae BAR Protein Gvp36 Possess Two Representative Conserved Motifs
3.2. NbSWP12 Forms Homodimer
3.3. NbSWP12 Binds to Ptdlns(3)P
3.4. NbSWP12 Targets to Yeast Cell Membrane and Endocytic Vesicle
3.5. NbSWP12 Rescues the Vesicle Genesis Defect of Yeast gvp36Δ
4. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Plasmid | Primer | Sequence |
---|---|---|
pCold I-Nbswp12 | swp12-F | CGGGATCCATGAAAGATTTTAAAAAGAA |
pUG35-Nbswp12 | swp12-R | GCGTCGACCTTAGTCCTCTCTAATGCTT |
pGADT7-Nbswp12 | 12AD-F | GGAATTCCATATGATGAAAGATTTTAAAAAGAAAATT |
12AD-R | CGCGGATCCTTACTTAGTCCTCTCTAATGCTTT | |
pGBKT7-Nbswp12 | 12BD-F | CGCGGATCC ATGAAAGATTTTAAAAAGAAAATT |
12BD-R | AAAACTGCAGTTACTTAGTCCTCTCTAATGCTTT |
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Chen, J.; Li, Z.; Sheng, X.; Huang, J.; Sun, Q.; Huang, Y.; Wang, R.; Wu, Y.; Long, M.; Bao, J.; et al. Heterologous Expressed NbSWP12 from Microsporidia Nosema bombycis Can Bind with Phosphatidylinositol 3-Phosphate and Affect Vesicle Genesis. J. Fungi 2022, 8, 764. https://doi.org/10.3390/jof8080764
Chen J, Li Z, Sheng X, Huang J, Sun Q, Huang Y, Wang R, Wu Y, Long M, Bao J, et al. Heterologous Expressed NbSWP12 from Microsporidia Nosema bombycis Can Bind with Phosphatidylinositol 3-Phosphate and Affect Vesicle Genesis. Journal of Fungi. 2022; 8(8):764. https://doi.org/10.3390/jof8080764
Chicago/Turabian StyleChen, Jie, Zhi Li, Xiaotian Sheng, Jun Huang, Quan Sun, Yukang Huang, Rong Wang, Yujiao Wu, Mengxian Long, Jialing Bao, and et al. 2022. "Heterologous Expressed NbSWP12 from Microsporidia Nosema bombycis Can Bind with Phosphatidylinositol 3-Phosphate and Affect Vesicle Genesis" Journal of Fungi 8, no. 8: 764. https://doi.org/10.3390/jof8080764