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 regulate d degradation and misfolded proteins for purposes of quality control destru ction. Ubiquitin ligases, or E3 proteins, promote ubiquitination by effecti ng the specific transfer of ubiquitin from the correct ubiquitin-conjugatin g enzyme, or E2 protein, to the target substrate. Substrate specificity is usually determined by specific sequence determinants, or degrons, in the ta rget substrate that are recognized by the ubiquitin ligase. In quality cont rol, 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 focus ed our attention on the first characterized quality control ubiquitin ligas e, the HRD complex, which is responsible for the endoplasmic reticulum (ER) -associated degradation (ERAD) of numerous ER-resident proteins. Using an i n vivo cross-linking assay, we directly examined the association of the sep arate HRD complex components with various ERAD substrates. We have discover ed that the HRD ubiquitin ligase complex associates with both ERAD substrat es and stable proteins, but only mediates ubiquitin-conjugating enzyme asso ciation with ERAD substrates. Our studies with the sterol pathway-regulated ERAD substrate Hmg2p, an isozyme of the yeast cholesterol biosynthetic enz yme HMG-coenzyme A reductase (HMGR), indicated that the HRD complex discern s between a degradation-competent "misfolded" state and a stable, tightly f olded state. Thus, it appears that the physiologically regulated, HRD-depen dent degradation of HMGR is effected by a programmed structural transition from a stable protein to a quality control substrate.