br Discussion The regulation of intracellular cholesterol
Discussion The regulation of intracellular cholesterol levels is a biological process of vital importance for cellular function and integrity, and involves robust transcriptional and post-transcriptional mechanisms that are sensitive to metabolic feedback control. Amongst post-transcriptional mechanisms, the ubiquitin proteasome system plays a prominent role as it allows rapid coupling of metabolic cues to fine tuning of metabolic flux through the mevalonate pathway. As such, the levels of the two rate-limiting biosynthetic angiotensin receptor blocker of the mevalonate pathway, HMGCR and SQLE, are subject to tight and well-coordinated regulation by the ubiquitin proteasome system [4,10], and are robustly degraded in response to sterol-derived cues. We recently identified the RING-type E3 ligase MARCH6 as the E3 responsible for the cholesterol-stimulated degradation of SQLE . In this study, by using a candidate-gene genetic approach, we now identify the E2 ubiquitin conjugating enzyme UBE2J2 as a partner of MARCH6 in cholesterol-stimulated degradation of SQLE in mammalian cells. Our finding that MARCH6 specifically uses UBE2J2 to promote SQLE degradation raises the intriguing question as to how this selectivity is achieved. One possibility is that expression of either MARCH6 or UBE2J2 is sensitive to the cellular sterol status, and that this may underlie their ability to act in concert to promote SQLE degradation. This would be akin to what we have previously reported for the E3 ubiquitin ligase Inducible degrader of the LDLR (IDOL), that promotes ubiquitylation of the LDLR in response to an increase in cellular sterol content and thus limits cholesterol uptake [28,29]. However, we show that, unlike SQLE, expression of both MARCH6 and UBE2J2 was refractory to manipulation of the cellular sterol status. As such, the determinants that govern the selective recruitment of UBE2J2 to MARCH6 for SQLE degradation remain to be determined. The issue of specificity within the ubiquitin proteasomal system extends beyond E2-E3 selective pairing and pertains also to the substrate specificity of E3 ubiquitin ligases. The ER-resident ligase MARCH6 is one out of several hundred E3 ubiquitin ligases present in mammalian cells . These ligases stimulate ubiquitylation of their targets in response to a plethora of signals. Importantly, the substrate specificity of some ligases may overlap, as evident in the case of ubiquitylation and degradation of HMGCR for which GP78, TRC8, and more recently RNF145, have been implicated [4,, , , , ]. Intriguingly, SQLE seems to be solely recognized by MARCH6 . The question of redundancy also extends to the E2 arm of the ubiquitin system. In contrast to the existence of multiple ERAD-associated E3s [30,31], only 3 ERAD-associated E2 ubiquitin conjugating enzymes are known . This implies that regulation of substrate degradation must also exist at the level of E2-E3 complex formation [13,32]. Indeed, we find that while degradation of HMGCR requires UBE2G2, as previously reported , that of SQLE relies predominantly on the use of UBE2J2. However, interestingly, UBE2G2KO cells also show a minor decrease in their ability to degrade SQLE in response to cholesterol. This may represent a vestigial function maintained in mammalian UBE2G2, as its yeast homolog Ubc7 acts as a promiscuous E2 ligase and interacts with gp78, Hrd1 and the yeast MARCH6-homologue Doa10 [20,33]. ERAD has been extensively studied in yeast and our findings highlight an additional divergence between yeast and mammalian ERAD. When comparing ERAD in yeast versus mammalian cells, a marked difference in complexity can be observed. While in yeast only three E3s (Hrd1, Asi and Doa10) and two E2s (Ubc6 and Ubc7) govern all ERAD , the array of E3 ligases is more diversified in mammalian cells, with up to 30 E3s, and 3 E2s [13,30]. While ERAD of the yeast homolog of SQLE, Erg1, is governed by Doa10 and the E2s Ubc6 and (promiscuous) Ubc7, our study demonstrates that UBE2J1 and UBE2J2, the two yeast Ubc6 homologues in mammalian cells, have distinct roles in ERAD of SQLE; Only Ube2J2 is implicated in SQLE degradation. Collectively, our findings shed more light on the physiological roles of ERAD E2s and underlines the importance of conducting more research to clarify the mechanisms by which ERAD contributes to the regulation of cholesterol homeostasis in mammalian cells.