Determinants of RING-E2 fidelity for Hrd1p, a membrane-anchored ubiquitin ligase.
A critical aspect of E3 ubiquitin ligase function is the selection of a particular E2 ubiquitin-conjugating enzyme to accomplish ubiquitination of a substrate. We examined the requirements for correct E2-E3 specificity in the RING-H2 ubiquitin ligase Hrd1p, an ER-localized protein known to use primarily Ubc7p for its function. Versions of Hrd1p containing the RING motif from homologous E3s were unable to carry out Hrd1p function, revealing a requirement for the specific Hrd1p RING motif in vivo. An in vitro assay revealed that these RING motifs were sufficient to function as ubiquitin ligases, but that they did not display the E2 specificity predicted from in vivo results. We further refined the in vitro assay of Hrd1p function by demanding not only ubiquitin ligase activity, but also specific activity that recapitulated both the E2 specificity and RING selectivity observed in vivo. Doing so revealed that correct E2 engagement by Hrd1p required the presence of portions of the Hrd1p soluble cytoplasmic domain outside the RING motif, the placement of the Hrd1p ubiquitin ligase in the ER membrane, and presentation of Ubc7p in the cytosolic context. We confirmed that these conditions supported the ubiquitination of Hrd1p itself, and the transfer of ubiquitin to the prototype substrate Hmg2p-GFP, validating Hrd1p self-ubiquitination as a viable assay of ligase function.
Bazirgan
Omar A
OA
Section of Cell and Developmental Biology, Division of Biological Sciences, University of California, San Diego, La Jolla, California 92093, USA.
Garza
Renee M
RM
Hampton
Randolph Y
RY
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002686:Chitin / Q000737:chemistry; D003600:Cytosol / Q000737:chemistry; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D047668:Inteins; D008051:Lipid Bilayers / Q000737:chemistry; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D025801:Ubiquitin / Q000737:chemistry; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry* / Q000502:physiology
D008051:Lipid Bilayers; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D002686:Chitin; D006639:Histidine; C077072:UBC7 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes; C104621:HRD1 protein, S cerevisiae; D044767:Ubiquitin-Protein Ligases