Ear1p and Ssh4p are new adaptors of the ubiquitin ligase Rsp5p for cargo ubiquitylation and sorting at multivesicular bodies.
The ubiquitylation of membrane proteins destined for the vacuole/lysosome is essential for their recognition by the endosomal sorting machinery and their internalization into vesicles of multivesicular bodies (MVBs). In yeast, this process requires Rsp5p, an essential ubiquitin ligase of the Nedd4 family. We describe here two redundant proteins, Ear1p and Ssh4p, required for the vacuolar targeting of several cargoes originating from the Golgi or the plasma membrane. Ear1p is an endosomal protein that interacts with Rsp5p through its PPxY motifs, and it is required for the ubiquitylation of selected cargoes before their MVB sorting. In-frame fusion of cargo to ubiquitin overcomes the need for Ear1p/Ssh4p, confirming a role for these proteins in cargo ubiquitylation. Interestingly, Ear1p is itself ubiquitylated by Rsp5p and targeted to the vacuole. Finally, Ear1p overexpression leads to Rsp5p accumulation at endosomes, interfering with some of its functions in trafficking. Therefore, Ear1p/Ssh4p recruit Rsp5p and assist it in its function at MVBs by directing the ubiquitylation of specific cargoes.
Léon
Sébastien
S
Institut Jacques Monod, Unité Mixte de Recherche 7592 Centre National de la Recherche Scientifique, Universités Paris 6 et 7, 75251 Paris, France. leon@ijm.jussieu.fr
Erpapazoglou
Zoi
Z
Haguenauer-Tsapis
Rosine
R
D033942:Adaptor Proteins, Vesicular Transport / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004705:Endocytosis; D056827:Endosomal Sorting Complexes Required for Transport; D011992:Endosomes / Q000378:metabolism*; D033704:Nucleotide Transport Proteins / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D043743:Ubiquitin-Protein Ligase Complexes / Q000378:metabolism*; D054875:Ubiquitination; D014617:Vacuoles / Q000378:metabolism
D033942:Adaptor Proteins, Vesicular Transport; C533265:EAR1 protein, S cerevisiae; D056827:Endosomal Sorting Complexes Required for Transport; C035528:FUR4 protein, S cerevisiae; D033704:Nucleotide Transport Proteins; C533266:SSH4 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D043743:Ubiquitin-Protein Ligase Complexes; C469287:RSP5 protein, S cerevisiae