Interaction of the Deubiquitinating Enzyme Ubp2 and the E3 Ligase Rsp5 Is Required for Transporter/Receptor Sorting in the Multivesicular Body Pathway

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.

Distinct ubiquitin ligases act sequentially for RNA polymerase II polyubiquitylation

“…1E Upper, compare lanes 1 and 2), supporting the idea that Ubp2 can also de-ubiquitylate RNAPII in vivo. As previously reported (24), ubp3 mutant cells also had an increased amount of ubiquitylated RNAPII ( Fig. 1E Upper, lane 3).…”

“…Therefore, it is formally possible that in vivo, Rsp5 normally only adds mono-ubiquitin. Interestingly, however, Rsp5 forms a complex with the ubiquitin protease Ubp2, which has been shown to affect certain other protein ubiquitylation pathways (23,24). We purified TAP-tagged Ubp2 and found that substoichiometric amounts of Rsp5 co-purified, as previously observed by others (23,24).…”

“…Experiments with ubp2⌬ cells support the idea that Rsp5 can add a K63 chain to RNAPII in vivo, but that this chain is trimmed by Ubp2. Rsp5 and Ubp2 interact (23,24), and our data with purified factors show that Ubp2 is capable of hydrolyzing K63 chains to produce mono-ubiquitylated RNAPII, further supporting the idea that Ubp2-mediated 'proofreading' of Rsp5 action occurs. We previously reported that Ubp3 plays a role in 'rescuing' RNAPII from degradation by reversing RNAPII ubiquitylation, and that it can hydrolyze K48-linked ubiquitin chains (25).…”

“…1E Lower, compare lanes 1 and 2), while increased levels of polyubiquitylated RNAPII were not observed in ubp3⌬ cells by this approach (Fig. 1E Lower, compare lanes 2 and 3), as predicted from its role in the rescue of RNAPII 'marked' for degradation by K48-linked polyubiquitin chains (24). Others have also shown that Ubp2 regulates K63-linked ubiquitin chain formation in vivo (27), so together these results suggest that Ubp2 has the ability to restrict RNAPII modification to mono-ubiquitylation by trimming K63-linked ubiquitin chains.…”

Synthetic Peptide Arrays for Pathway-Level Protein Monitoring by Liquid Chromatography-Tandem Mass Spectrometry

“…Yeast (strain S288C) soluble cell-free extract was generated by glass bead beating as described previously (23). 5 mg (500 l of 10 mg/ml protein) of soluble extract was precipitated overnight at Ϫ20°C by the addition of 5 volumes of ice-cold acetone.…”