| Description |
Multivesicular bodies (MVBs) are late endosomal compartments that contain numerous intralumenal vesicles (ILVs) enriched in specific proteins and lipids. The ILVs are delivered to the vacuolar lumen when MVBs fuse with the vacuole. The ILVs form by invagination of the limiting membrane of the endosome along with the cargo. ILV production is catalyzed by the concerted action of proteins complexes called Endosomal Sorting Complexes Required for Transport (ESCRTs). ESCRTs are subdivided into functionally distinct complexes called ESCRT-0, -I, -II, -III and the Vps4 complex. Deletion analyses and biochemical studies suggest their sequential order of function, i.e. ESCRT-0 recruits ESCRT-I, which in turn activates ESCRT-II and so on. The predominant signal for cargo protein recognition/selection by ESCRTs seems to be ubiquitination. The early-acting ESCRT complexes (-0 and -I) are considered to sort cargo while late-acting ESCRT-III and Vps4 remodel membrane for ILV production. ESCRT-II links cargo sorting with ILV production by initiating assembly of ESCRT-III in a manner that is somehow regulated by early-acting ESCRTs. Cargo proteins are deubiquitinated by Doa4 just before ILV formation to recycle ubiquitin for further rounds of cargo ubiquitination. Several questions remain regarding cargo sorting into ILVs. Is ubiquitin an essential sorting signal for cargo proteins? Should cargo proteins bind the early-acting ESCRTs to be efficiently sorted into ILVs? Details about the arrangement and stoichiometry of ESCRTs and regulation of `late-acting' ESCRT-III proteins by `early-acting' ESCRTs remain unclear. Here, we directly address these questions by using fluorescence microscopy, protein biochemistry and molecular biology tools in Saccharomyces cerevisiae. We report that binding to any ESCRT complex can mediate protein sorting into the vacuole and that ubiquitin is not the only signal that can mediate this process. We provide mechanistic insights into sorting of cytosolic proteins into ILVs. We also report a role for deubiquitination in efficient release of ubiquitinated cargo proteins into ILVs. We have identified a constitutively active mutant of ESCRT-II that bypasses the need for ESCRT-0 or ESCRT-I, suggesting that these early ESCRTs are not essential and have redundant functions. Using the active mutant of ESCRT-II, we decoupled ESCRT-III assembly from early ESCRT-mediated cargo sorting. We report that activation of ESCRT-III alone is not sufficient for ILV formation, suggesting an essential role for cargo to form ILVs. |
