Structural control of endoplasmic reticulum-associated degradation: effect of chemical chaperones on 3-hydroxy-3-methylglutaryl-CoA reductase.

The endoplasmic reticulum (ER) quality control pathway destroys misfolded and unassembled proteins in the ER. Most substrates of this ER-associated degradation (ERAD) pathway are constitutively targeted for destruction through recognition of poorly understood structural hallmarks of misfolding. However, the normal yeast ER membrane protein 3-hydroxy-3-methylglutaryl-CoA reductase (Hmg2p) undergoes ERAD that is physiologically regulated by sterol pathway signals. We have proposed that Hmg2p ERAD occurs by a regulated transition to an ERAD quality control substrate. Consistent with this, we had previously shown that Hmg2p is strongly stabilized by chemical chaperones such as glycerol, which stabilize misfolded proteins. To understand the features of Hmg2p that permit regulated ERAD, we have thoroughly characterized the effects of chemical chaperones on Hmg2p. These agents caused a reversible, immediate, direct change in Hmg2p degradation consistent with an effect on Hmg2p structure. We devised an in vitro limited proteolysis assay of Hmg2p in its native membranes. In vitro, chemical chaperones caused a dramatic, rapid change in Hmg2p structure to a less accessible form. As in the living cell, the in vitro action of chemical chaperones was highly specific for Hmg2p and completely reversible. To evaluate the physiological relevance of this model behavior, we used the limited proteolysis assay to examine the effects of changing in vivo degradation signals on Hmg2p structure. We found that changes similar to those observed with chemical chaperones were brought about by alteration of natural degradation signal. Thus, Hmg2p can undergo significant, reversible structural changes that are relevant to the physiological control of Hmg2p ERAD. These findings support the idea that Hmg2p regulation is brought about by regulated alteration of folding state. Considering the ubiquitous nature of quality control pathways in biology, it may be that this strategy of regulation is widespread.