Human homologs of Ubc6p ubiquitin-conjugating enzyme and phosphorylation of HsUbc6e in response to endoplasmic reticulum stress.
Ubiquitin-conjugating enzyme Ubc6p is a tail-anchored protein that is localized to the cytoplasmic face of the endoplasmic reticulum (ER) membrane and has been implicated in the degradation of many misfolded membrane proteins in yeast. We have undertaken characterization studies of two human homologs, hsUbc6 and hsUbc6e. Both possess tail-anchored protein motifs, display high conservation in their catalytic domains, and are functional ubiquitin-conjugating enzymes as determined by in vitro thiol-ester assay. Both also display induction by the unfolded protein response, a feature of many ER-associated degradation (ERAD) components. Post-translational modification involving phosphorylation of hsUbc6e was observed to be ER-stress-related and dependent on signaling of the PRK-like ER kinase (PERK). The phosphorylation site was mapped to Ser-184, which resides within the uncharacterized region linking the highly conserved catalytic core and the C-terminal transmembrane domain. Phosphorylation of hsUbc6e also did not alter stability, subcellular localization, or interaction with a partner ubiquitin-protein isopeptide ligase. Assays of hsUbc6e(S184D) and hsUbc6e(S184E), which mimic the phosphorylated state, suggest that phosphorylation may reduce capacity for forming ubiquitin-enzyme thiol-esters. The occurrence of two distinct Ubc6p homologs in vertebrates, including one with phosphorylation modification in response to ER stress, emphasizes diversity in function between these Ub-conjugating enzymes during ERAD processes.
Oh
Ray S
RS
Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8; Program in Genetics & Genomic Biology, The Hospital for Sick Children, Toronto, Ontario M5G 1L7, Canada.
Bai
Xinli
X
Program in Genetics & Genomic Biology, The Hospital for Sick Children, Toronto, Ontario M5G 1L7, Canada.
Rommens
Johanna M
JM
Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8; Program in Genetics & Genomic Biology, The Hospital for Sick Children, Toronto, Ontario M5G 1L7, Canada. Electronic address: jrommens@sickkids.ca.
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D025801:Ubiquitin / Q000737:chemistry; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry* / Q000502:physiology*; D019892:eIF-2 Kinase / Q000737:chemistry / Q000502:physiology*
D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D044763:Ubiquitin-Conjugating Enzymes; D019892:eIF-2 Kinase; C082461:UBC6 protein, S cerevisiae; C489967:UBE2J1 protein, human