The ubiquitin ligase Rsp5p is required for modification and sorting of membrane proteins into multivesicular bodies.
Precursor forms of vacuolar proteins with transmembrane domains, such as the carboxypeptidase S Cps1p and the polyphosphatase Phm5p, are selectively sorted in endosomal compartments to vesicles that invaginate, budding into the lumen of the late endosomes, resulting in the formation of multivesicular bodies (MVBs). These proteins are then delivered to the vacuolar lumen following fusion of the MVBs with the vacuole. The sorting of Cps1p and Phm5p to these structures is mediated by ubiquitylation, and in doa4 mutant cells, which have reduced level of free ubiquitin, these proteins are missorted to the vacuolar membrane. A RING-finger ubiquitin ligase Tul1p has been shown to participate in the ubiquitylation of Cps1p and Phm5p. We show here that the HECT-ubiquitin ligase Rsp5p is also required for the ubiquitylation of these proteins, and therefore for their sorting to MVBs. Rsp5p is an essential ubiquitin ligase containing an N-terminal C2 domain followed by three WW domains, and a C-terminal catalytic HECT domain. In cells with low levels of Rsp5p (npi1 mutant cells), vacuolar hydrolases do not reach the vacuolar lumen and are instead missorted to the vacuolar membrane. The C2 domain and both the second and third WW domains of Rsp5p are important determinants for sorting to MVBs. Ubiquitylation of Cps1p was strongly reduced in the npi1 mutant strain and ubiquitylation was completely abolished in the npi1 tul1Delta double mutant. These data demonstrate that Rsp5p plays a novel and key role in intracellular trafficking, and extend the currently very short list of substrates ubiquitylated in vivo by several different ubiquitin ligases acting cooperatively.
Morvan
Joelle
J
Institut Jacques Monod, CNRS-UMRC9922, Universités Paris 6 and Paris 7-Denis Diderot, 2 place Jussieu, 75251-Paris-cedex 05, France.
Froissard
Marine
M
Haguenauer-Tsapis
Rosine
R
Urban-Grimal
Danièle
D
D002268:Carboxypeptidases / Q000378:metabolism; D056827:Endosomal Sorting Complexes Required for Transport; D008565:Membrane Proteins / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism; D043743:Ubiquitin-Protein Ligase Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014617:Vacuoles / Q000378:metabolism*
D056827:Endosomal Sorting Complexes Required for Transport; D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D043743:Ubiquitin-Protein Ligase Complexes; D002268:Carboxypeptidases; C019836:CPS1 protein, S cerevisiae; C469287:RSP5 protein, S cerevisiae