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The ubiquitin ligase Rsp5p is required for modification and sorting of membrane proteins into multivesicular bodies
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International audience. Precursor forms of vacuolar proteins with transmem-brane domains, such as the carboxypeptidase S Cps1pand the polyphosphatase Phm5p, are selectively sortedin endosomal compartments to vesicles that invaginate,budding into the lumen of the late endosomes, resultingin the formation of multivesicular bodies (MVBs). Theseproteins are then delivered to the vacuolar lumen follow-ing fusion of the MVBs with the vacuole. The sorting ofCps1p and Phm5p to these structures is mediated byubiquitylation, and in doa4 mutant cells, which havereduced level of free ubiquitin, these proteins are misso rtedto the vacuolar membrane. A RING-finger ubiquitin ligaseTul1p has been shown to part icipate in the ubiquitylati on ofCps1p and Phm5p. We show here that the HECT-ubiquit inligase Rsp5p is also required for the ubiquitylation of theseproteins, and therefore for their sorting to MVBs. Rsp5p isan essential ubiquitin ligase containing an N-terminal C2domain followed by three WW domains, and a C-terminalcatalytic HECT domain. In cells with low levels of Rsp5p(npi1 mutant cells), vacuolar hydrolases do not reach thevacuolar lumen and are instead missorted to the vacuolarmembrane. The C2 domain and both the second and thirdWW domains of Rsp5p are important determinants forsorting to MVBs. Ubiquitylation of Cps1p was strong lyreduced in the npi1 mutant strain and ubiquitylation wascompletely abolished in the npi1 tul1D double mutant.These data demonstrate that Rsp5p plays a novel and keyrole in intracellular trafficking, and extend the currently veryshort list of substrates ubiquitylated in vivo by severaldifferent ubiquitin ligases acting cooperatively.
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