Ste6p Mutants Defective in Exit from the Endoplasmic Reticulum (ER) Reveal Aspects of an ER Quality Control Pathway inSaccharomyces cerevisiae

Abstract

We are studying the intracellular trafficking of the multispanning membrane protein Ste6p, the a-factor transporter in Saccharomyces cerevisiae and a member of the ATPbinding cassette superfamily of proteins. In the present study, we have used Ste6p as model for studying the process of endoplasmic reticulum (ER) quality control, about which relatively little is known in yeast. We have identified three mutant forms of Ste6p that are aberrantly ER retained, as determined by immunofluorescence and subcellular fractionation. By pulse-chase metabolic labeling, we demonstrate that these mutants define two distinct classes. The single member of Class I, Ste6 -166p, is highly unstable. We show that its degradation involves the ubiquitin-proteasome system, as indicated by its in vivo stabilization in certain ubiquitin-proteasome mutants or when cells are treated with the proteasome inhibitor drug MG132. The two Class II mutant proteins, Ste6 -13p and Ste6 -90p, are hyperstable relative to wild-type Ste6p and accumulate in the ER membrane. This represents the first report of a single protein in yeast for which distinct mutant forms can be channeled to different outcomes by the ER quality control system. We propose that these two classes of ER-retained Ste6p mutants may define distinct checkpoint steps in a linear pathway of ER quality control in yeast. In addition, a screen for high-copy suppressors of the mating defect of one of the ER-retained ste6 mutants has identified a proteasome subunit, Hrd2p/p97, previously implicated in the regulated degradation of wild-type hydroxymethylglutaryl-CoA reductase in the ER membrane.

Previously unknown role for the ubiquitin ligase Ubr1 in endoplasmic reticulum-associated protein degradation

“…Hrd1/Der3 is thought to act mainly in concert with the E2s Ubc1 and Ubc7, whereas Doa10 mainly works together with Ubc6 and Ubc7 (14,29,30). Canonical ERAD of the substrate Ste6* depends on Doa10; therefore, Ubc6 and Ubc7 are the preferred ubiquitinconjugating enzymes in this process (20). In Ubr1-dependent ERAD, the combined deletion of UBC6 and UBC7 or combined deletion of UBC1 and UBC7 showed a small but measurable decline of Ste6* degradation ( Fig.…”

“…Because Ste6* contains a misfolded domain exposed to the cytosol (17,18,20), recognition of this substrate by the cytosolic ligase Ubr1 seems plausible. Therefore, we tested whether in the absence of canonical ligases, ERAD substrates that carry their misfolded domain within the ER lumen also would be degraded via the Ubr1-dependent ERAD route.…”

“…To address this hypothesis, we selected the misfolded polytopic membrane protein Ste6* as a model substrate. Ste6* is a C-terminally truncated version of the transporter of the mating type a pheromone and is retained in the ER membrane as a result of the exposition of its misfolded domain to the cytosol (17,20). In cells lacking the two canonical ubiquitin ligases, Ste6* is not fully stabilized but remains a target for degradation (17,19).…”

Pbn1p: An essential endoplasmic reticulum membrane protein required for protein processing in the endoplasmic reticulum of budding yeast

“…Ste6p* is a C-terminal cytosolic truncation mutant of this multispanning membrane protein that is retained in the ER and subsequently degraded by the ERAD machinery (29). Although the lesion in Ste6p* is cytosolic, we determined whether lack of Pbn1p affected the ERAD of this protein.…”

“…Although the lesion in Ste6p* is cytosolic, we determined whether lack of Pbn1p affected the ERAD of this protein. We harvested WT and GAL-PBN1 cells bearing a plasmid containing STE6*-HA (29,30) and assayed for ERAD of this protein by pulse-chase analysis. At 60 min of chase, both WT cells and cells lacking Pbn1p showed complete degradation of Ste6p*-HA (Fig.…”