Evidence supporting a major role for the ubiquitin-proteasome pathway in sterol-accelerated degradation of reductase was first provided by the observation that proteasome inhibition blocks the process 18
, leading to the accumulation of ubiquitinated forms of reductase on ER membranes 8
. This ubiquitination is obligatory for degradation of reductase and exhibits an absolute requirement for the presence of Insigs 37, 38
. Reduction of Insig-1 and Insig-2 mRNA by genetic mutation or RNAi-mediated knockdown abrogates sterol-dependent ubiquitination of endogenous reductase, rendering the enzyme refractory to accelerated degradation. Moreover, sterol-induced ubiquitination of reductase exhibits an absolute Insig requirement in transient transfection assays. Mutation of the YIYF sequence in reductase, which blocks Insig binding, prevents regulated ubiquitination and slows the enzyme’s degradation. In contrast, conservative substitutions of arginine for lysines 89 and 248 in the membrane domain of reductase () do not block Insig binding, but the substitutions rather abolish ubiquitination and subsequent degradation of reductase. Thus, lysines 89 and 248 in reductase are implicated as sites for Insig-mediated, sterol-induced ubiquitination. It is important to note that mutation of lysines 89 and 248 blocks ubiquitination and degradation of reductase in the context of the full-length enzyme, suggesting that the catalytic domain does not contribute to ubiquitination. This is consistent with the observation that the soluble catalytic domain is dispensable for sterol-regulated degradation 16
How might Insig binding impart recognition of reductase by the ubiquitinating machinery? This question was addressed by examining reductase ubiquitination in a permeabilized cell system 39
. Sterol-depleted cells were permeabilized with low concentrations of the mild detergent digitonin such that nearly all of the cytosolic proteins were released into the supernatant upon centrifugation, whereas membrane proteins such as reductase remained associated with the pellet fraction. The pellet of permeabilized cells supports Insig-dependent ubiquitination of reductase that is stimulated by additions of ATP, sterols, and rat liver cytosol in vitro
. Surprisingly, reductase ubiquitination is potently stimulated by oxygenated derivatives of cholesterol, including 24-, 25-, and 27-hydroxycholesterol, but not by cholesterol itself. The significance of this finding will be discussed in more detail below.
Ubiquitination of proteins is a multistep process, involving the action of at least three types of enzymes 40
. In the first step, ubiquitin is activated by the ubiquitin-activating enzyme (E1), which forms a thiol ester between a reactive cysteine residue in E1 and the C-terminus of ubiquitin. Next, ubiquitin is transferred from E1 to a catalytic cysteine of the ubiquitin-conjugating enzyme (E2) thiol ester. The third type of enzyme, ubiquitin ligase (E3) facilitates transfer of activated ubiquitin from E2 to a lysine residue in the substrate (or a previously attached ubiquitin). Once a poly-ubiquitin chain of sufficient size is built, the substrate is recognized and subsequently degraded by proteasomes. Only two E1 enzymes exist, and both are cytosolic proteins. In contrast, a variety of E2s and E3s, both soluble and membrane-bound have been described 41
. The exquisite sensitivity of substrate ubiquitination is ultimately determined by the E3, either alone or in combination with its cognate E2.
Fractionated S100 from Hela cells was utilized to determine which component of the reductase ubiquitinating machinery (E1, E2 and E3) is provided by rat liver cytosol in the permeabilized cell system 39
. These fractions were first described by Hershko and co-workers 42, 43
and were generated by separating Hela cell S100 into fractions that bind (Fraction II) or do not bind (Fraction I) an anion exchange resin. It was subsequently determined that Fraction I contained ubiquitin, whereas Fraction II contains E1 44
. Fraction II effectively replaces rat liver cytosol for regulated ubiquitination of reductase in permeabilized cells, but Fraction I does not. Immunodepletion of E1 eliminates the reductase ubiquitinating activity of rat liver cytosol. Consistent with this, purified E1 replaces rat liver cytosol for sterol-regulated ubiquitination of reductase in permeabilized cells. Thus, E1 is the only cytosolic protein required for reductase ubiquitination, which indicates the reductase E2 and E3 are membrane-associated proteins. This notion is consistent with the localization of apparent sites of reductase ubiquitination, lysines 89 and 248, which are cytosolically exposed and are predicted to lie immediately adjacent to transmembrane helices three and seven ().
Results from the analysis of reductase ubiquitination in permeabilized cells indicated that Insig binding results in recruitment of enzymes that ubiquitinate reductase. Coimmunoprecipitation experiments, coupled with tandem mass spectroscopy, were utilized to identify membrane proteins that associate with the sterol-dependent reductase-Insig complex. These studies revealed that Insig-1 binds to a known membrane-anchored ubiquitin ligase called gp78 45
. The cDNA for gp78 predicts a 643-amino acid protein that can be divided into four domains. The N-terminal domain of 298 amino acids contains five to seven membrane-spanning helices that anchors the protein to ER membranes and mediates association with Insig-1. The membrane attachment region of gp78 is followed by a 43-amino acid region with a RING finger consensus sequence that confers ubiquitin ligase activity 46
. Following the RING domain is a 42-amino acid region homologous to Cue1p, an ER membrane protein in yeast that serves as a membrane anchor for Ubc7p, a cytosolic ubiquitin-conjugating enzyme 47
. Recently, this region of gp78 has been shown to directly bind to Ufd1, a cytosolic protein that modulates gp78 ubiquitin ligase activity, thereby enhancing ubiquitination and degradation of the enzyme’s substrates 48
. Finally, the extreme C-terminus of gp78 (48 amino acids) mediates an interaction with VCP (Valosin-containing protein, also known as p97), an ATPase that has been implicated in the post-ubiquitination steps of ER-associated degradation (ERAD) 49
At least three lines of evidence indicate that gp78, through its binding to Insig-1, initiates sterol-accelerated degradation of reductase. 1) Overexpression of the membrane domain of gp78 blocks Insig-mediated, sterol-accelerated degradation of reductase, suggesting that the membrane domain of gp78 competes with full-length gp78 for binding to Insig-1, thereby abolishing reductase ubiquitination. 2) Sterols trigger binding of gp78 to reductase in an Insig-dependent, sterol-regulated manner. The specificity of this binding is illustrated by the inability of gp78 to bind Scap, regardless of the presence or absence of sterols and/or Insigs. 3) RNAi-mediated knockdown of gp78 prevents sterol-regulated ubiquitination and degradation of endogenous reductase. Importantly, the effect of gp78 knockdown is specific inasmuch as knockdown of a related membrane-bound ubiquitin ligase, Hrd1, does not effect reductase ubiquitination. Another function of gp78, besides its role as a ubiquitin ligase, is to couple ubiquitination of reductase to degradation through its association with VCP. Indeed, coimmunoprecipitation experiments show that gp78 is an intermediary in association of VCP and Insig-1. Moreover, knockdown of VCP by RNAi prevents sterol-accelerated degradation of endogenous reductase and a dominant-negative ATPase-deficient mutant of VCP blocks sterol-regulated degradation of transfected reductase.
The identification of gp78 as an E3 ubiquitin ligase that mediates reductase ubiquitination has important implications for yet another mode of sterol regulation. The regulation of Insig-1 contrasts that of reductase in that Insig-1 becomes ubiquitinated and is rapidly degraded by proteasomes in sterol-depleted
. Ubiquitination of Insig-1 is mediated by gp78 51
. When sterols induce reductase to bind Insig-1, ubiquitination is diverted toward reductase and the enzyme becomes rapidly degraded. However, when sterols cause Scap to bind Insig-1, gp78 is displaced and no longer ubiquitinates Insig-1, thereby stabilizing the protein. This reaction helps to explain why reductase is degraded when it binds to Insig-1, whereas Scap binding to Insig-1 leads to retention in the ER. In addition, gp78-mediated ubiquitination and degradation of Insig-1 provides a mechanism for a recently appreciated process termed “convergent feedback inhibition” 50
. In sterol-depleted cells, Scap-SREBP complexes no longer bind Insig-1, which in turn becomes ubiquitinated and degraded. Thus, Scap-SREBP complexes are free to exit the ER and translocate to the Golgi, where the SREBPs are processed to the nuclear form that stimulates transcription of target genes, including the Insig-1 gene. Increased transcription of the Insig-1 gene leads to increased synthesis of Insig-1 protein, but the protein is ubiquitinated and degraded until sterols build up to levels sufficient to trigger Scap binding. Thus, inhibition of SREBP processing requires convergence of newly synthesized Insig-1 and newly acquired sterols.