Randolph Y. Hampton
The ER Degradation Pathway
We are broadly interested in protein degradation as a mode of biological regulation. Selective protein degradation underlies many processes of biological and medical interest including the cell cycle, immune signaling, metabolic control, developmental decisions, and cellular responses to stress. Currently our focus is on a well-known but poorly understood substrate of regulated degradation, the rate-limiting enzyme of cholesterol synthesis, HMG-CoA reductase (HMG-R; (1)). HMG-R is subject to regulated degradation as part of cellular control of cholesterol synthesis, such that the degradation of the protein is altered to attempt to balance enzyme levels with cellular need for the many essential products of the cholesterol pathway. Since the degradation of HMG-R is regulated in this manner, the study of HMG-R degradation holds the combined promise of revealing generally important molecular mechanisms of selective protein degradation, and the unknown, clinically relevant processes used by cells to measure and modulate the synthesis of cholesterol.
We study regulated degradation of the HMG-R using a combination of genetic, cell biological, molecular biological, and biochemical approaches (2-4). The discovery that the HMG-R protein of the baker's yeast is regulated at the level of degradation has opened the door to a complete genetic analysis of the turnover mechanism and the signaling pathway that couples it to the cholesterol pathway (1). We have defined a new class of genes, known as HRD (pronounced "herd") genes, that are responsible for HMG-CoA Reductase Degradation in yeast. The collection of genes so far includes both novel (but conserved) proteins, and familiar components of known cellular degradation machinery (2). Our working model is that a subset of HRD-encoded proteins act specifically on HMG-R and related substrates to bring about degradation by the action of the broadly employed, essential complex known as the 26S proteasome. Furthermore, our data indicate that a separate group of genes are involved in measuring the cholesterol pathway and coupling that information to the HRD-mediated degradation of HMG-R. The genes are referred to as CRD ("curd") genes, for Control of Reductase Degradation. As the entire ensemble of CRD/HRD genes are isolated, our picture will become more refined and mechanistic. We have tested this hypothesis by designing a screen specifically targeted to that class of crd mutants that can not slow the degradation of Hmg2p. At present we have isolated two complementation groups of crd mutants in a pilot screen that is far from saturation.
In parallel with these genetic studies, we are discovering the features of the HMG-R protein that allow it to undergo degradation in a specific, programmed manner. Recalling that the cell is ~50% protein, one can see that the sequence or structural features that allow selection of a single protein for degradation are exceedingly important, and in most cases, unknown.
Finally, we have recently found that a specific molecular event, the addition of the small protein ubiquitin to the HMG-R molecule, appears to be a central determinant of the degradation process (3). In addition we have found that the ubiquitination of HMG-R is regulated in a manner consistent with the known specificity and regulation of HMG-R stability. These observations will allow a clear set of molecular tests for the mechanisms of action of the HRD and CRD genes. Furthermore, clarification of the molecular mechanism of HMG-R degradation is a sine qua non for the discovery of agents that modulate this regulatory axis in the clinical setting.