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The identification and characterization of new components of the multivesicular body (MVB) sorting pathway


The Multivesicular Body (MVB) Sorting pathway provides a mechanism for the delivery of transmembrane proteins into the lumen of the vacuole/lysosome for degradation. It has been shown that the MVB sorting pathway plays a critical role in biological processes including receptor downregulation and retroviral budding. Studies by the Emr lab and others have demonstrated that ubiquitin modification acts in cis as a signal for the sorting of cargo into this pathway. Using a genetic selection designed to identify mutants that missort MVB cargoes, we isolated the ubiqutin ligase Rsp5. This selection identified a point mutation in ubiquitin ligase Rsp5 (Rsp5 -326). At the permissive temperature, this mutant is specifically defective for ubiquitination and sorting of the ubiquitin-dependent MVB cargo pCPS (precursor Carboxypeptidase S) but not ligand-induced ubiquitination of Ste2. Thus, Rsp5 plays an essential role in the ubiquitination and targeting of cargo proteins into the MVB pathway. The ESCRT complexes associated proteins are required for the sorting of cargo into the MVB pathway. While the crystal structures of two ESCRT complexes have have been determined, the molecular mechanisms underlying the assembly and regulation of the ESCRT machinery is are still poorly understood. Here, we identify a new component of the ESCRT-I complex, Mvb12, and demonstrate that Mvb12 binds to the coiled-coil domain of the ESCRT-I subunit Vps23. We show that ESCRT-I adopts an oligomeric state in the cytosol, the formation of which requires the coiled- coil domain of Vps23 as well as Mvb12. Loss of Mvb12 results in the disassembly of the ESCRT-I oligomer and the formation of a stable complex of ESCRT-I and ESCRT-II in the cytosol. We propose that Mvb12 stabilizes stabilizes ESCRT-I in an oligomeric, inactive state in the cytosol to ensure that the ordered recruitment and assembly of ESCRT- I and -II is spatially and temporally restricted to the surface of the endosome, following activation of the MVB sorting reaction

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