Role of 26S proteasome and HRD genes in the degradation of 3-hydroxy-3-methylglutaryl-CoA reductase, an integral endoplasmic reticulum membrane protein.

Abstract

3-hydroxy-3-methylglutaryl-CoA reductase (HMG-R), a key enzyme of sterol synthesis, is an integral membrane protein of the endoplasmic reticulum (ER). In both humans and yeast, HMG-R is degraded at or in the ER. The degradation of HMG-R is regulated as part of feedback control of the mevalonate pathway. Neither the mechanism of degradation nor the nature of the signals that couple the degradation of HMG-R to the mevalonate pathway is known. We have launched a genetic analysis of the degradation of HMG-R in Saccharomyces cerevisiae using a selection for mutants that are deficient in the degradation of Hmg2p, an HMG-R isozyme. The underlying genes are called HRD (pronounced "herd"), for HMG-CoA reductase degradation. So far we have discovered mutants in three genes: HRD1, HRD2, and HRD3. The sequence of the HRD2 gene is homologous to the p97 activator of the 26S proteasome. This p97 protein, also called TRAP-2, has been proposed to be a component of the mature 26S proteasome. The hrd2-1 mutant had numerous pleiotropic phenotypes expected for cells with a compromised proteasome, and these phenotypes were complemented by the human TRAP-2/p97 coding region. In contrast, HRD1 and HRD3 genes encoded previously unknown proteins predicted to be membrane bound. The Hrd3p protein was homologous to the Caenorhabditis elegans sel-1 protein, a negative regulator of at least two different membrane proteins, and contained an HRD3 motif shared with several other proteins. Hrdlp had no full-length homologues, but contained an H2 ring finger motif. These data suggested a model of ER protein degradation in which the Hrdlp and Hrd3p proteins conspire to deliver HMG-R to the 26S proteasome. Moreover, our results lend in vivo support to the proposed role of the p97/TRAP-2/Hrd2p protein as a functionally important component of the 26S proteasome. Because the HRD genes were required for the degradation of both regulated and unregulated substrates of ER degradation, the HRD genes are the agents of HMG-R degradation but not the regulators of that degradation.

ERAD‐dependent control of the Wnt secretory factor Evi

“…To identify E3 ligases involved in Evi degradation, we used siRNAs to screen a selected panel of E3s implicated previously in ERAD or predicted to reside in the ER membrane (Neutzner et al , 2011; Figs 6C and EV5A). Unexpectedly, depletion of E3s with well‐characterized roles in ERAD such as Hrd1 (Hampton et al , 1996) and gp78 (Fang et al , 2001) was not sufficient to stabilize Evi. Instead, Evi was stabilized upon knockdown of CGRRF1, a relatively uncharacterized ER‐resident E3 ligase that has not yet been linked to ERAD (Figs 6C and EV5C).…”

“…E3 ligases confer specificity of ubiquitin conjugation by interacting with the substrates to promote efficient ubiquitin transfer. While some ER‐resident E3s like Hrd1 (Hampton et al , 1996; Bernasconi et al , 2010; Kanehara et al , 2010; Christianson et al , 2011) seem to accommodate a broad range of substrates, others appear to be more selective including TRC8 (Stagg et al , 2009) and RNF170 (Lu et al , 2011). We identified the RING‐finger protein CGRRF1 as an Evi‐binding E3 ligase that modulates the abundance of Evi.…”

Cellular response to endoplasmic reticulum stress: a matter of life or death

“…Ubiquitin conjugating enzymes participating in ERAD include Ubc1p, Ubc6p, Ubc7p and its membrane-anchored binding partner Cue1p. [50][51][52] Other proteins physically and genetically interact with the ubiquitin-conjugating machinery during dislocation, including Der1p, Der3p/Hrd1p and Hrd3p, [53][54][55][56] although the exact biochemical functions of all these components in yeast are unknown. In addition to signaling for degradation, ubiquitination may also assist in removing some substrates from the ER via a ratchet mechanism, whereby polyubiquitinated chains are prevented from passively sliding backwards through the dislocation channel.…”

Proteins of the endoplasmic-reticulum-associated degradation pathway: domain detection and function prediction

“…The molecular function of Hrd3p remains elusive, although its sequence shows localized similarity to C. elegans SEL-1 [49], a negative regulator of LIN-12 [50], over approx. 40 amino acids.…”