title: Distinct machinery is required in Saccharomyces cerevisiae for the endoplasmic reticulum-associated degradation of a multispanning membrane protein and a soluble luminal protein.
abstract: The folding and assembly of proteins in the endoplasmic reticulum (ER) lumen and membrane are monitored by ER quality control. Misfolded or unassembled proteins are retained in the ER and, if they cannot fold or assemble correctly, ultimately undergo ER-associated degradation (ERAD) mediated by the ubiquitin-proteasome system. Whereas luminal and integral membrane ERAD substrates both require the proteasome for their degradation, the ER quality control machinery for these two classes of proteins likely differs because of their distinct topologies. Here we establish the requirements for the ERAD of Ste6p*, a multispanning membrane protein with a cytosolic mutation, and compare them with those for mutant form of carboxypeptidase Y (CPY*), a soluble luminal protein. We show that turnover of Ste6p* is dependent on the ubiquitin-protein isopeptide ligase Doa10p and is largely independent of the ubiquitin-protein isopeptide ligase Hrd1p/Der3p, whereas the opposite is true for CPY*. Furthermore, the cytosolic Hsp70 chaperone Ssa1p and the Hsp40 co-chaperones Ydj1p and Hlj1p are important in ERAD of Ste6p*, whereas the ER luminal chaperone Kar2p is dispensable, again opposite their roles in CPY* turnover. Finally, degradation of Ste6p*, unlike CPY*, does not appear to require the Sec61p translocon pore but, like CPY*, could depend on the Sec61p homologue Ssh1p. The ERAD pathways for Ste6p* and CPY* converge at a post-ubiquitination, pre-proteasome step, as both require the ATPase Cdc48p. Our results demonstrate that ERAD of Ste6p* employs distinct machinery from that of the soluble luminal substrate CPY* and that Ste6p* is a valuable model substrate to dissect the cellular machinery required for the ERAD of multispanning membrane proteins with a cytosolic mutation.
authors.affiliation: Department of Cell Biology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
authors.forename: Gregory
authors.initials: G
authors.lastname: Huyer
authors.forename: Wachirapon F
authors.initials: WF
authors.lastname: Piluek
authors.forename: Zoya
authors.initials: Z
authors.lastname: Fansler
authors.forename: Stefan G
authors.initials: SG
authors.lastname: Kreft
authors.forename: Mark
authors.initials: M
authors.lastname: Hochstrasser
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Brodsky
authors.forename: Susan
authors.initials: S
authors.lastname: Michaelis
mesh_terms: D000251:Adenosine Triphosphatases; D043402:Cathepsin A / Q000737:chemistry; D018797:Cell Cycle Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000378:metabolism; D008954:Models, Biological; D009097:Multienzyme Complexes / Q000378:metabolism; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013997:Time Factors; D025801:Ubiquitin / Q000378:metabolism; D000074405:Valosin Containing Protein
chemical_list: D018797:Cell Cycle Proteins; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D009097:Multienzyme Complexes; D010455:Peptides; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D043402:Cathepsin A; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D000251:Adenosine Triphosphatases; C095973:SSA1 protein, S cerevisiae; C000614177:CDC48 protein, S cerevisiae; D000074405:Valosin Containing Protein