title: An unusual transmembrane helix in the endoplasmic reticulum ubiquitin ligase Doa10 modulates degradation of its cognate E2 enzyme.
abstract: In the endoplasmic reticulum (ER), nascent membrane and secreted proteins that are misfolded are retrotranslocated into the cytosol and degraded by the proteasome. For most ER-associated degradation (ERAD) substrates, ubiquitylation is essential for both their retrotranslocation and degradation. Yeast Doa10 is a polytopic membrane ubiquitin ligase (E3) that along with its cognate ubiquitin-conjugating enzymes (E2s), Ubc7 and the C-terminally membrane-anchored Ubc6, makes a major contribution to ER-associated degradation. Ubc6 is also a substrate of Doa10. One highly conserved Doa10 element, the uncharacterized ~130-residue TEB4-Doa10 domain, includes three transmembrane helices (TMs). We find that the first of these, TM5, includes an absolutely conserved ΦPΦXXG motif that is required for Doa10 function, as well as highly conserved negatively charged glutamate and aspartate residues. The conservative exchange of the TM5 glutamate to aspartate (doa10-E633D) results in complete stabilization of Ubc6 but has little if any effect on other substrates. Unexpectedly, mutating the glutamate to glutamine (doa10-E633Q) specifically accelerates Ubc6 degradation by ~5-fold. Other substrates are weakly stabilized in doa10-E633Q cells, consistent with reduced Ubc6 levels. Notably, catalytically inactive ubc6-C87A is degraded in doa10-E633Q but not wild-type cells, but an active version of Ubc6 is required in trans. Fusion of the Ubc6 TM to a soluble protein yields a protein that is degraded in a doa10-E633Q-dependent manner, whereas fusion of the C-terminal TM from an unrelated protein does not. These results suggest that the TEB4-Doa10 domain regulates Doa10 association with the Ubc6 membrane anchor, thereby controlling the degradation rate of the E2.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8114, USA. stefan.kreft@uni-konstanz.de
authors.forename: Stefan G
authors.initials: SG
authors.lastname: Kreft
authors.forename: Mark
authors.initials: M
authors.lastname: Hochstrasser
mesh_terms: D019943:Amino Acid Substitution; D004721:Endoplasmic Reticulum / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability / Q000502:physiology; D020125:Mutation, Missense; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*
chemical_list: D029701:Saccharomyces cerevisiae Proteins; C082461:UBC6 protein, S cerevisiae; C077072:UBC7 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes; C091362:SSM4 protein, S cerevisiae; D044767:Ubiquitin-Protein Ligases