In vivo action of the HRD ubiquitin ligase complex: mechanisms of endoplasmic reticulum quality control and sterol regulation.
Ubiquitination is used to target both normal proteins for specific regulated degradation and misfolded proteins for purposes of quality control destruction. Ubiquitin ligases, or E3 proteins, promote ubiquitination by effecting the specific transfer of ubiquitin from the correct ubiquitin-conjugating enzyme, or E2 protein, to the target substrate. Substrate specificity is usually determined by specific sequence determinants, or degrons, in the target substrate that are recognized by the ubiquitin ligase. In quality control, however, a potentially vast collection of proteins with characteristic hallmarks of misfolding or misassembly are targeted with high specificity despite the lack of any sequence similarity between substrates. In order to understand the mechanisms of quality control ubiquitination, we have focused our attention on the first characterized quality control ubiquitin ligase, the HRD complex, which is responsible for the endoplasmic reticulum (ER)-associated degradation (ERAD) of numerous ER-resident proteins. Using an in vivo cross-linking assay, we directly examined the association of the separate HRD complex components with various ERAD substrates. We have discovered that the HRD ubiquitin ligase complex associates with both ERAD substrates and stable proteins, but only mediates ubiquitin-conjugating enzyme association with ERAD substrates. Our studies with the sterol pathway-regulated ERAD substrate Hmg2p, an isozyme of the yeast cholesterol biosynthetic enzyme HMG-coenzyme A reductase (HMGR), indicated that the HRD complex discerns between a degradation-competent "misfolded" state and a stable, tightly folded state. Thus, it appears that the physiologically regulated, HRD-dependent degradation of HMGR is effected by a programmed structural transition from a stable protein to a quality control substrate.
Gardner
R G
RG
Section of Cell and Developmental Biology, Division of Biology, University of California, San Diego, La Jolla, California 92093, USA.
Shearer
A G
AG
Hampton
R Y
RY
D000818:Animals; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D005656:Fungal Proteins / Q000378:metabolism; D005990:Glycerol / Q000494:pharmacology; D006903:Hydroxymethylglutaryl CoA Reductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008025:Ligases / Q000235:genetics / Q000378:metabolism*; D008148:Lovastatin / Q000494:pharmacology; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D011485:Protein Binding / Q000187:drug effects*; D011487:Protein Conformation; D017510:Protein Folding; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D013261:Sterols / Q000096:biosynthesis; D013379:Substrate Specificity; D014233:Tricarboxylic Acids / Q000494:pharmacology; D014357:Trypsin / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases
D019086:Bridged Bicyclo Compounds, Heterocyclic; C109280:CUE1 protein, S cerevisiae; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D005656:Fungal Proteins; C104622:HRD3 protein, S cerevisiae; D007527:Isoenzymes; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins; D013261:Sterols; D014233:Tricarboxylic Acids; C075117:squalestatin 1; D008148:Lovastatin; D006903:Hydroxymethylglutaryl CoA Reductases; C079437:UBE2L3 protein, human; D044763:Ubiquitin-Conjugating Enzymes; C104621:HRD1 protein, S cerevisiae; D044767:Ubiquitin-Protein Ligases; D014357:Trypsin; D008025:Ligases; D005990:Glycerol