Mechanisms of tether function in endolysosomal trafficking - Renewal - Resubmission 01

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Project Summary The accurate trafficking of proteins through membrane compartments depends on the ability of specific vesicles to recognize one another and undergo efficient fusion. Accuracy is conferred in part by molecular tethers, which are multivalent complexes that bind to vesicle determinants and foster specific vesicle-vesicle contacts. Endosome-lysosome trafficking is required for the processing of cargo taken up from the cell surface, and also paradoxically for the formation of some secretory organelles. Two key tethers involved in endolysosomal trafficking are CORVET (class C core vacuole/endosome tethering) and HOPS (homotypic fusion and protein sorting). Both are hetero-hexameric complexes, whose importance in cell and organismal physiology is shown by the fact that human variants in the genes encoding CORVET/HOPS subunits are linked with mucopolysaccharidosis, renal dysfunction, neurodegeneration and cancer, among other diseases. At the cellular level, CORVET and HOPS both function in homotypic fusion, i.e., allow two vesicles with the same Rab determinants to recognize one another. The mechanisms of action of CORVET/HOPS have been deduced primarily via in vitro reconstitution experiments. However, key aspects of current models have yet to be tested in vivo, including important mechanistic details such as whether the complexes are stably associated with membranes, whether individual complexes undergo cyclical assembly/disassembly, and whether different subunits have distinct cycling dynamics. In addition, the assembly state of tethers on vesicles is unknown, a salient question because the tether subunits are structurally similar to coat proteins that form large assemblies on membranes. In the lineage of ciliates including the model organism Tetrahymena thermophila, the HOPS complex was lost. Concurrently, CORVET complexes multiplied in these cells, with individual complexes becoming specialized for distinct endolysosomal pathways. Due to its specific evolutionary history combined with experimental strengths, Tetrahymena offers a unique new system to analyze these universal tethers. Issues to be addressed include the copy number of CORVET complexes associated with vesicles, the dynamics of both the holo-complexes and individual subunits, and the role of specific protein-protein interactions. Experiments will be based on a combination of biochemical analysis, correlated light and electron microscopy to follow tagged proteins expressed at endogenous levels, and cell fusion to detect protein dynamics. Correlative light and electron microscopy will be used to visualize the ultrastructure of membrane compartments decorated by tagged proteins, allowing for detailed in vivo analysis of tether function.