Browsing by Subject "Endosom"

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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.
Intrazelluläres Trafficking des intestinalen Anionenaustauschers Down-Regulated in Adenoma (DRA;SLC26A3)
(2011) Lissner, Simone; Graeve, Lutz
Electroneutral NaCl absorption occurs from the small intestine to the distal colon. This ion exchange is preferentially mediated by DRA and NHE3. Knockout mice, which suffer from chronic diarrhea, as well as the human genetic disorder congenital chloride diarrhea, in which a nonfunctional DRA leads to life-threatening diarrhea emphasize the importance of these two transporters. To elucidate this defective NaCl absorption it is necessary to understand the physiological regulation of these two transport proteins within enterocytes as well as the responsible extra- and intracellular signal transduction pathways. Both transport proteins interact with PDZ adaptor proteins of the NHERF family. Furthermore, both exchangers are partially localized within lipid rafts. The situation for NHE3 is complex in that its lipid raft localization is not only necessary for its normal activity but also for its basal and stimulated trafficking. Lipid rafts are involved in PI3-kinase dependent exocytosis of NHE3. Since the function of NHE3 and DRA appears to be regulated in parallel the function of DRA maybe depends on its rafts association as well. Thus the first objective of this thesis was to investigate whether the lipid raft association of DRA is essential for the surface expression and transport activity of DRA and also to analyze whether DRA is inserted into the plasma membrane in a PI3-kinase and lipid raft dependent manner. The present data show that: (A) Disruption of lipid raft integrity leads to functional inhibition and decreased cell surface expression of DRA. In HEK cells the inhibition of DRA activity as well as the decreased cell surface expression are entirely dependent on the presence of the PDZ interaction motif of DRA. In Caco-2/BBE cells on the other hand only part of the inhibition of DRA activity by disruption of raft integrity depends on the ability of DRA to interact with PDZ adaptor proteins. (B) Basal activity as well as basal surface expression of DRA depend on PI3-kinase activity in a way that requires the ability of DRA to interact with PDZ adaptor proteins. (C) Lipid rafts and PI3-kinase are situated along the same pathway, where DRA is present in lipid rafts before it is inserted into the plasma membrane. However, the inhibition of PI3-kinase has no influence on the raft association of DRA. Furthermore, the disruption of raft integrity does not inhibit the PI3-kinase activity. Based on these findings a model can be established as follows: DRA is present in lipid rafts in an intracellular fraction. Insertion into the plasma membrane from this intracellular compartment requires the interaction with one (or several) PDZ adaptor proteins, raft integrity and the action of PI3-kinase. To characterize the interplay between PI3-kinase, raft association and PDZ interaction of DRA with its insertion into the plasma membrane the recycling pathway of DRA was then investigated. The generated data show that the proteolytic degradation of DRA-ETKFminus occurs faster than the degradation of wild type DRA. Endosomal distribution of DRA depends on its PDZ-binding motif. The sorting process from early to recycling endosomes depends on the interaction of DRA with one or several PDZ adaptor proteins. Expression of dominant negative Rab11a leads to a decreased surface expression and transport activity of DRA. In conclusion, it was shown in this thesis that an intense interplay between PDZ interaction, lipid raft association, PI3-kinase and the activity and surface expression of DRA exists. It was also shown that the endosomal distribution of DRA depends on its PDZ-binding motif. Finally, it was demonstrated that DRA is recycled to the plasma membrane by Rab11a-enriched recycling endosomes.