Abstract
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.
Highlights
Protein ubiquitination is essential for the proper functioning of many eukaryotic cellular processes
An aberrant accumulation of membrane proteins at the plasma membrane can result from a defect in multivesicular bodies (MVBs) formation, as has been reported with ESCRT mutants [12]
Further evidence that places Ubp2 at the MVB sorting step is the fact that sensitivity to 5-FU does not seem to worsen in a vps37Dubp2D double mutant when compared to a vps37D single mutant alone
Summary
Protein ubiquitination is essential for the proper functioning of many eukaryotic cellular processes. While the covalent conjugation of polyubiquitin chains to a protein by ubiquitin ligases often leads to subsequent targeting and degradation by the 26S proteasome [1], ubiquitin attachment has been shown to be critical for the correct execution of non-proteasomal events, ranging from the regulated methylation of histones during transcriptional elongation [2] to the intracellular trafficking of proteins [3]. For the latter, ubiquitination serves as a key signal mediating the internalization, intracellular transport and subsequent recycling or vacuolar degradation of plasma membranebound receptors and transporters [4]. It undergoes Rsp5-dependent ubiquitination, a modification crucial for its sorting into MVBs [14], followed by vacuolar degradation after fusion of MVBs with the vacuole
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