Interaction of the deubiquitinating enzyme Ubp2 and the e3 ligase Rsp5 is required for transporter/receptor sorting in the multivesicular body pathway.
Protein ubiquitination is essential for many events linked to intracellular protein trafficking. We sought to elucidate the possible involvement of the S. cerevisiae deubiquitinating enzyme Ubp2 in transporter and receptor trafficking after we (this study) and others established that affinity purified Ubp2 interacts stably with the E3 ubiquitin ligase Rsp5 and the (ubiquitin associated) UBA domain containing protein Rup1. UBP2 interacts genetically with RSP5, while Rup1 facilitates the tethering of Ubp2 to Rsp5 via a PPPSY motif. Using the uracil permease Fur4 as a model reporter system, we establish a role for Ubp2 in membrane protein turnover. Similar to hypomorphic rsp5 alleles, cells deleted for UBP2 exhibited a temporal stabilization of Fur4 at the plasma membrane, indicative of perturbed protein trafficking. This defect was ubiquitin dependent, as a Fur4 N-terminal ubiquitin fusion construct bypassed the block and restored sorting in the mutant. Moreover, the defect was absent in conditions where recycling was absent, implicating Ubp2 in sorting at the multivesicular body. Taken together, our data suggest a previously overlooked role for Ubp2 as a positive regulator of Rsp5-mediated membrane protein trafficking subsequent to endocytosis.
Lam
Mandy H Y
MH
Banting and Best Department of Medical Research, Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada.
Urban-Grimal
Danièle
D
Bugnicourt
Amandine
A
Greenblatt
Jack F
JF
Haguenauer-Tsapis
Rosine
R
Emili
Andrew
A
D000483:Alleles; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism; D004705:Endocytosis; D010450:Endopeptidases / Q000378:metabolism*; D056827:Endosomal Sorting Complexes Required for Transport; D005472:Fluorouracil / Q000494:pharmacology; D015966:Gene Expression Regulation, Fungal; D000072235:Mating Factor; D008957:Models, Genetic; D033704:Nucleotide Transport Proteins / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport / Q000235:genetics; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D043743:Ubiquitin-Protein Ligase Complexes / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism
D056827:Endosomal Sorting Complexes Required for Transport; C035528:FUR4 protein, S cerevisiae; D033704:Nucleotide Transport Proteins; D010455:Peptides; D029701:Saccharomyces cerevisiae Proteins; D000072235:Mating Factor; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases; D010450:Endopeptidases; C059237:ubiquitin-Nalpha-protein hydrolase; C469287:RSP5 protein, S cerevisiae; D005472:Fluorouracil