Browsing by Subject "ESCRT"

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Influence of the newly identified Mos10 interaction partner Vps68on ESCRT-III function
(2021) Alsleben, Sören; Kölling, Ralf
The endosomal sorting complex required for transport (ESCRT) is a part of the heteromeric complex machinery consisting of ESCRT-0, -I, -II, and -III ensuring functional protein traffic of endocytic and biosynthetic cargo. Stepwise sorting of labeled cargo material inside the lumen of the endosome by invagination and abscission of the endosomal membrane to form intraluminal vesicles (ILV’s) is mediated by the ESCRT-III complex. The complex consists of eight members of which Vps20, Snf7, Vps2, and Vps24 are considered ESCRT-III essential subunits, and Chm7, Did2, Ist1, and Mos10/Vps60 are commonly labeled as complex associated proteins. The correct interplay between the proteins ensures cargo sorting into the MVB (multivesicular body) pathway and transport from the late endosome into the vacuolar lumen for degradation. Besides the initial function of vacuolar protein sorting (vps), the complex is involved in a multitude of cellular processes like cell abscission, virus budding, autophagy, and remaining nuclear envelope integrity. The step-wise assembly of the ESCRT-III complex is mediated after the cascade-like ESCRT-0 to ESCRT-II complex formation at the membrane budding site, collecting cargo protein for invagination into the endosomal lumen. ESCRT-III Vps20 is recruited to the membrane by the ESCRT-II member Vps25, then nucleating Snf7 association and oligomerization. Additional assembly of ESCRT-III members like Vps24 and Vps2 further drives membrane bending away from the cytosol to the final abscission event, before being recycled back to cytosolic monomers by Vps4. Although Mos10 has been implicated in the recycling step of the ESCRT-III units by interacting with the Vps4/Vta1 complex, the protein’s function remains poorly characterized. This thesis tried to find new insights in Mos10 functionality by finding yet uncharacterized interacting partners, thus connecting the protein to new putative non-endosomal functions or understanding its role in the established ESCRT-III complex. For this purpose, a series of crosslinking experiments with tagged variants of Mos10 were performed. Purification was achieved by IMAC (Immobilized Metal Ion Affinity Chromatography) after adding a poly-his sequence to the protein and by immunoprecipitation of sfGFP tagged Mos10. Both methods revealed a multitude of putative Mos10 interacting partners by MS analysis to be further reduced by applying the SILAC (stable isotope labeling with amino acids in cell culture) technique. After selecting possible Mos10 interacting partners, IP and Co-IP experiments of tagged candidate variants were used to identify an interaction between the two proteins. An interaction between Mos10-6His and Vps68-13myc besides native Mos10 and Vps68-fGFP could be verified by purification of Vps68 and co-precipitating Mos10. The influence of Vps68 on the assembly and composition of the ESCRT-III complex was examined. After Vps68 depletion, an enrichment of the core subunits Snf7, Vps2, and Vps24 in the complex was detected with a reduced number of Did2, Ist1, and Mos10 molecules. Thus, it appears that ESCRT-III disassembly is blocked in ∆vps68 mutant. The influence of VPS68 deletion on the intracellular localization of ESCRT-III proteins was examined by fluorescence microscopy with sfGFP-tagged variants. While the localization of most ESCRT-III proteins was not significantly altered, a marked relocalization was observed for Mos10. In wildtype, Mos10-sfGFP was localized at the vacuolar membrane, while in ∆vps68 it was dispersed into vesicular structures enriched at the cell cortex. Further, the impact of VPS68 deletion on the sorting of the endocytic cargo protein Ste6 was investigated. By cycloheximide chase experiments, it could be shown that Ste6 is strongly stabilized in a ∆vps68 mutant. This indicates that the transport of the protein to the yeast vacuole for degradation is blocked. The ∆vps68 block in endocytic trafficking was compared with other mutants of the vps-pathway, whose site of action has been established. These experiments show that the VPS68 deletion neither leads to a class D phenotype, as in ∆vps21, nor to a class E phenotype, as in ∆snf7. The Ste6-GFP distribution in the ∆vps68 mutant rather resembles wildtype with more pronounced accumulation of endosomal dots. The data taken together suggest that Vps68 acts after the formation of the ESCRT-III complex and is required for cargo delivery from the late endosome to the vacuolar lumen.