Sorting of yeast late endosomal SNAREs and assembly of this SNARE complex
Zusammenfassung der Projektergebnisse
Eukaryotic cells are characterized by membrane bound organelles, which are connected by vesicular transport. Cargo has to be sorted into membrane buds. Vesicles are pinched off by adaptor proteins and coat proteins from the membrane buds. These vesicles fuse with the target membrane with the help of SNARE proteins on both membranes via complexes of their SNARE motifs. We are interested in the transport between trans Golgi network (TGN), endosomes and lysosomes, which are called vacuoles in yeast. These pathways have a crucial role in degradation. The baker’s yeast Saccharomyces cerevisiae is a valuable model system to analyze eukaryotic cell function. We are interested in the yeast SNARE Vti1p, which functions in transport from the TGN to late endosomes, fusion with the vacuole and transport from early endosomes back to the TGN as part of different SNARE complexes. During this project we found that unfolding of the N-terminal domain of Vti1p in a temperature-sensitive mutant results in defects in these transport steps and lower levels of Vti1p-containing SNARE complexes. However, deletion of N-terminal domain of Vti1p did not result in these phenotypes indicating that cells can adapt to the permanent loss of this domain. We collaborated with a group from Hefei (China), who solved the structure of the N-terminal domain of Vti1p in a complex with the ENTH domain of Ent3p. Ent3p functions in formation of vesicles at the TGN or endosome and recruits Vti1p into budding vesicles as cargo adaptor. We demonstrated the importance of residues in the binding site for interaction and localization of Vti1p in living cells by generation and analysis of point mutations. The ENTH domain protein was identified as cargo adaptor for the SNARE Tlg2, which functions in transport from early endosomes to the TGN. Analyzing vacuolar delivery of the plasma membrane protein Can1p-GFP we found different phenotypes for cells lacking Ent3p or Ent5p. Cells without Ent3p accumulated Can1p-GFP in endosomes indicating that transport from endosomes to the vacuole is blocked. Can1p-GFP was transported faster to vacuoles in the absence of Ent5p, indicating that Ent5p is required for transport from endosomes back to the TGN. We found that the uncharacterized protein Scy1p interacted with Ent3p and Ent5p. Defects due to the absence of Scy1p were not detected. Synthetic defects were observed in ent3∆ ent5∆ scy1∆ cells providing evidence for a functional connection between Scy1p, Ent3p and Ent5p.
Projektbezogene Publikationen (Auswahl)
- (2011) ENTH domains bind on opposite sides of two SNAREs. Proc. Natl. Acad. Sci. USA 108, 12277-12282
Wang, J., Gossing, M., Fang, P., Zimmermann, J., Li, X., Fischer von Mollard, G., Niu, L., Teng, M.
- (2013) Importance of the N-terminal domain of the Qb-SNARE Vti1p for different membrane transport steps in the yeast endosomal system. PLoS ONE 8, e66304
Gossing, M., Chidambaram, S., Fischer von Mollard, G.
(Siehe online unter https://doi.org/10.1371/journal.pone.0066304)
