STE6P MUTANTS DEFECTIVE IN EXIT FROM THE ENDOPLASMIC-RETICULUM (ER) REVEAL ASPECTS OF AN ER QUALITY-CONTROL PATHWAY IN SACCHAROMYCES-CEREVISIAE

We are studying the intracellular trafficking of the multispanning mem brane protein Ste6p, the a-factor transporter in Saccharomyces cerevis iae and a member of the ATP-binding cassette superfamily of proteins. In the present study, we have used Ste6p as model for studying the pro cess of endoplasmic reticulum (ER) quality control, about which relati vely little is known in yeast. We have identified three mutant forms o f Ste6p that are aberrantly ER retained, as determined by immunofluore scence and subcellular fractionation. By pulse-chase metabolic labelin g, 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. Th e two Class II mutant proteins, Ste6-13p and Ste6-90p, are hyperstable relative to wild-type Ste6p and accumulate in the ER membrane. This r epresents the first report of a single protein in yeast for which dist inct mutant forms can be channeled to different outcomes by the ER qua lity 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 s uppressors 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 r eductase in the ER membrane.