INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Ubiquitin-mediated, proteasome-dependent degradation is often employed by the cell to destroy both normal and misfolded proteins for purposes of regulation and quality control (39, 51). In general, proteins destined for degradation by the proteasome are covalently modified with the small protein ubiquitin through the collective action of a hierarchy of enzymes (21, 40, 46). A single ubiquitin-activating enzyme, or E1 protein, activates ubiquitin for transfer to a small number of ubiquitin-conjugating enzymes, or E2 proteins, which are divergent in their targeting function and underlie the first level of specificity for substrate selection. The ubiquitin-conjugating enzymes then covalently attach the ubiquitin moiety to the side chain of an internal lysine residue within their target substrates (11, 14, 79), in a reaction requiring additional specificity factors referred to as ubiquitin ligases, or E3 proteins, which constitute the largest class of proteins in the ubiquitination hierarchy.

Currently known ubiquitin ligases have catalytic subunits that fall into two distinct classes: HECT domain proteins, such as E6-AP and Rsp5p (35, 43, 74), and RING-H2 domain proteins, such as Rbx1p and Hrd1p (3, 32, 76, 78). HECT domain ubiquitin ligase action involves transient covalent linkage of ubiquitin to the ubiquitin ligase itself, followed by transfer of the attached ubiquitin moiety to the target substrate (75). RING-H2 domain ubiquitin ligase action appears to involve promotion of direct ubiquitin transfer between the ubiquitin-conjugating enzyme and the substrate. In addition to catalyzing substrate ubiquitination, many ubiquitin ligases also catalyze their own ubiquitination (3, 18, 27, 57, 61, 67).

Current models suggest that RING-H2 ubiquitin ligases function by providing specific binding sites for both the target substrate and the relevant ubiquitin-conjugating enzyme to effect transfer of ubiquitin from the conjugating enzyme to the target. In fact, the RING-H2 domain of cCbl mediates direct interaction with UbcH7 (92). The simultaneous binding of the substrate and ubiquitin-conjugating enzyme to the RING-H2 ubiquitin ligase to stimulate substrate ubiquitination can be referred to as a mutual binding mechanism. The specific binding of target substrates may be brought about directly by the ubiquitin ligase itself or by proteins associated with the ubiquitin ligase. For example, in the SCF ubiquitin ligase complex, the RING-H2 protein Hrt1p (also called Rbx1p or Roc1p) employs a scaffold complex containing an effector protein that specifically binds to the target substrate (19, 62, 76-78). Different substrate-binding proteins are incorporated into the scaffold complex to change the complex's substrate specificity (19, 23, 63, 77). Similarly, the APC ubiquitin ligase complex, containing the RING-H2 protein Apc11p (53, 62, 76, 90), also utilizes a scaffold complex to bind the target substrate (52, 64, 89, 90). In contrast, the N-end rule ubiquitin ligase contains multiple substrate recognition sites within the RING-H2 protein itself (1, 2, 30, 70), allowing the same protein to bind multiple substrates and related allosteric regulators. Other ubiquitin ligases that also directly bind their specific substrate targets include cCbl (47, 54) and Mdm2 (41, 42). In each case, the specificity of the ubiquitin ligase complex is a consequence of its ability to specifically bind the target protein.

Ubiquitin-mediated degradation is employed in cellular quality control to remove misfolded and misassembled proteins (51, 84). For quality control degradation to be effective, the ubiquitination machinery must recognize common structural hallmarks of damage or misfolding in a diverse group of proteins with little or no primary sequence homology. Recently, we and others have identified a RING-H2 ubiquitin ligase complex, referred to as the HRD complex below, that is responsible for degradation of numerous quality control substrates (3, 6, 27, 32, 67). Presumably, these and other quality control ubiquitin ligases also function by promoting association between the substrate and the ubiquitin-conjugating enzyme. However, it is not clear if the mutual binding model of ubiquitin ligase function is applicable to the large and varied set of possible quality control substrates with no sequence similarity. It may be that the quality control ubiquitin ligases recognize appropriate substrates by evaluation of the folding state rather than specific sequence motifs within the target, although no evidence has yet been presented for such a model.

A significant component of cellular quality control degradation occurs by endoplasmic reticulum (ER)-associated degradation (ERAD). Mutant lumenal or integral membrane proteins incapable of attaining correct structure or proper assembly are destroyed by ERAD (4, 20, 32, 36, 45, 66, 68, 82, 85, 87, 88, 93). ERAD is not restricted to aberrant proteins, as a significant fraction of newly made, normal proteins are destroyed by ERAD as part of normal ER physiology (55, 80, 83). Furthermore, ERAD is employed in the feedback regulation of HMG-coenzyme (CoA) reductase (HMGR), so that signals from the sterol synthesis pathway control HMGR stability (12, 17, 25, 31, 34, 60).

In Saccharomyces cerevisiae, ERAD is mediated in large part by the action of a RING-H2 ubiquitin ligase complex composed of the integral ER membrane proteins Hrd1p (Der3p) and Hrd3p (3, 6, 27, 32, 66, 67). Hrd1p contains an N-terminal, multispanning membrane anchor and a C-terminal cytosolic domain (27), which contains a RING-H2 motif homologous to several characterized ubiquitin ligases (18, 47, 49, 57). Consistent with this homology, Hrd1p functions both in vitro and in vivo as a ubiquitin ligase (3), catalyzing the processive transfer of ubiquitin to itself and other proteins. In vitro, Hrd1p shows preference for a misfolded protein as a multiubiquitination substrate (3) and thus may directly participate in some aspects of quality control recognition. In vivo, Hrd1p is rate limiting for ubiquitination and degradation of numerous ERAD substrates (3, 27, 67) and specifically employs only Ubc7p and Ubc1p in its action (3), with Ubc7p being quantitatively more important in the ERAD pathway. Hrd3p is a single-spanning ER membrane protein, with the majority of its sequence residing in the ER lumen (27, 32, 67, 71). The Hrd3p lumenal regions are both necessary and sufficient for Hrd3p action in ERAD (27). One function of the Hrd3p lumenal domain is to regulate the activity and stability of the cytosolic Hrd1p RING-H2 domain through transmembrane communication mediated by the Hrd1p membrane anchor (27). However, Hrd3p has ERAD functions independent of Hrd1p stabilization (27).

Because Hrd1p and Hrd3p form an ER membrane-associated ubiquitin ligase complex required for quality control ERAD, it is likely that the complex recognizes common structural features among its diverse substrates. Here, we directly tested the model that in vivo HRD complex promotes association between ERAD substrates and the ER ubiquitin-conjugating enzyme Ubc7p. From these studies, it is clear that the HRD complex does indeed mediate fruitful proximity between ERAD substrates and the appropriate ubiquitin-conjugating enzyme. Additionally, it appears that the HRD complex scans ER proteins for their degradation status and, upon encountering a protein that appears to be misfolded, promotes access of the appropriate ubiquitin-conjugating enzyme to the substrate, allowing robust yet selective ubiquitination of the desired target. According to this model, and in contrast to regulatory ubiquitin ligases, the HRD complex does not promote specificity by restricting its interactions only to substrates. Rather, the HRD complex functions in ubiquitination by discerning substrate properties subsequent to interaction. This model also suggests a mechanism for regulated ERAD of HMGR, which provides a significant component of eukaryotic regulation of the sterol pathway. Regulated HMGR ERAD appears to occur through a controlled structural transition of the HMGR transmembrane domain that allows it to be recognized as a quality control substrate by the HRD machinery. These studies have mechanistic implications for understanding both the specificity of quality control degradation and the clinically relevant axis of sterol regulation that employs ERAD to control the cellular synthesis of cholesterol (29).

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Materials and reagents. All enzymes were obtained from New England Biolabs (Beverly, Mass.). Chemical reagents were obtained from Sigma Chemical (St. Louis, Mo.). Dithiosuccinimidylproprionate (DSP) was obtained from Pierce (Rockford, Ill.). Protein A-Sepharose CL-4B was obtained from Amersham Pharmacia (Piscataway, N.J.). Lovastatin, L-659,699, and zaragozic acid were generously donated by Merck (Rahway, N.J.). ECL enhanced chemiluminescence immunodetection reagents were from Amersham Corp. (Arlington Heights, Ill.). Anti-Myc 9E10 antibody was used as a cell culture supernatant obtained by growing the 9E10 hybridoma (ATCC CRL 1729) in RPMI 1640 culture medium (Life Technologies, Grand Island, N.Y.) with 10% fetal calf serum. Antihemagglutinin (HA) antibody was an ascites fluid obtained from Babco (Berkeley, Calif.). Affinity-purified goat anti-mouse immunoglobulin-horseradish peroxidase (HRP) conjugate was obtained from Sigma Chemical.

Recombinant DNA and molecular cloning. The splicing-by-overlap-extension method was use to make epitope-tagged versions of each gene (38). PCR was performed as previously described (24). A list of primers used is available upon request. All epitope-tagged plasmids were verified for their ability to completely complement a null mutation in their respective genomic copy.

Strains and media. Escherichia coli DH5 strains were grown at 37°C in Luria-Bertani medium with ampicillin (100 µg/ml). Yeast strains were grown at 30°C in minimal medium supplemented with glucose and the appropriate amino acids, as described (24). The lithium acetate method was used to transform yeast cells with plasmid DNA (44).

MAT his3200 lys2-801 ade2-101 leu2 ura3-52

MAT his3200 lys2-801 ade2-101 leu2 ura3-52

Ubiquitination assays. Ubiquitination assays were performed as previously described (3).

Degradation assay. Cycloheximide-chase assays were performed as previously described (24).

Cross-linking assays. Cross-linking assays were done as previously described (27), except that 30 µl of anti-HMGR antiserum was used for the immunoprecipitation. In a given set of experiments, samples were always run on a single gel so that transfer, immunoreactivity, and exposure times were kept constant between samples that were to be compared.

Flow cytometry. Flow cytometry was performed as previously described (24), using a FACSscan (Beckton Dickinson, Palo Alto, Calif.) analytical flow microfluorimeter with settings for fluorescein-labeled antibody analysis. To examine the effects of lovastatin and zaragozic acid on Hmg2p-green fluorescent protein (GFP) steady-state levels, the drugs were added to early-log-phase cultures (optical density at 600 nm [OD₆₀₀] of <0.2) to the desired final concentrations, and the cells were grown for an additional 4 h (two doublings). In some cases, cells were grown to log phase, with 10% glycerol initially added to the medium or 10% glycerol added at the time of drug addition.

TLC. Thin-layer chromatography (TLC) was performed as previously described (28).

Protease protection assays. Microsomes were isolated as previously described (27). Protease protection assays were performed as previously described (27) except that anti-Hmg2p antiserum was used to detect Hmg2p.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

ERAD of numerous ER lumenal and membrane proteins requires the Hrd1p/Hrd3p ubiquitin ligase complex (4, 6, 66, 67), including the regulated ERAD of the yeast HMGR isozyme Hmg2p (3, 27, 32). In addition to Hrd1p and Hrd3p, ERAD requires the ubiquitin-conjugating enzyme Ubc7p (4, 34, 37, 66) and its membrane association factor Cue1p (5). From this, it seems likely that some or all of the HRD complex proteins and their associated factors could directly interact with substrates to target them for ubiquitination.

To test this hypothesis, we required an assay that would allow detection of the transient interactions that might exist between the ER membrane-bound HRD ubiquitin ligase complex components and the targeted degradation substrates. Furthermore, the substrates and HRD complex under study are, for the most part, integral membrane proteins, and the assay had to be amenable to the physical constraints inherent with such molecules. Finally, to study the participation of the HRD complex in regulation of Hmg2p, the assay had to allow for the presence and action of small signaling molecules from the mevalonate pathway that our studies show are at low cellular abundance and transiently produced (25, 28, 31). Assays normally used for interaction studies are the yeast two-hybrid assay, coimmunoprecipitation, and chemical cross-linking. To measure transient interactions, however, the two-hybrid assay was not a viable option because by nature, the transient interactions would likely be of low affinity, nor is there a transparent adaptation of this technique for studying interactions between membrane proteins. Similarly, because the HRD complex components and some of its substrates are integral membrane proteins (27, 31, 32), a cross-linking approach was required in lieu of the more usual coimmunoprecipitation studies of ubiquitin ligase activity and substrate association (47, 49, 78, 86). Coimmunoprecipitation assays were not applicable to our system, since the solubilization methods required to extract the proteins from the ER membrane would likely disrupt the interactions between substrates and membrane-bound HRD proteins and would most certainly dilute or remove the transient signaling molecules that control ERAD of Hmg2p. In contrast, chemical cross-linking has been used with great success to determine transient interactions between components of the ER membrane-bound Sec61p translocon and its translocation substrates (48, 58, 59, 65, 72). Additionally, we developed an in vivo modification of the cross-linking assay (27) which allows the signaling molecules that control Hmg2p ERAD to remain and function at normal levels and location. Although cross-linking between two proteins indicates their close proximity and is therefore informative of potential protein-protein interactions, the absence of cross-linking does not necessarily preclude an interaction. With this caveat, we proceeded with the cross-linking studies to test interactions between the HRD complex and its substrates.

To analyze substrate-HRD complex interactions, we used the in vivo chemical cross-linking assay previously developed to examine interactions between the subunits of the HRD complex itself (27). For ease of detection, we used functional, HA epitope-tagged versions of each protein in the HRD complex, which have been described previously (3, 27, 71). In addition, we employed a uniquely available set of ERAD substrates from our studies of regulated degradation of yeast HMGR (26, 32), each with an added myc epitope tag. These included the mevalonate pathway-regulated Hmg2p, the homologous but constitutively stable Hmg1p, and the constitutively degraded 6myc-Hmg2p.

Specific cross-linking of Ubc7p with ERAD substrates. The current model for RING-H2 ubiquitin ligase function is that the ligase promotes proximity between the ubiquitin-conjugating enzyme and the target substrate through their mutual binding, which leads to direct ubiquitination of the substrate. However, ubiquitin ligase-mediated proximity of ubiquitin- conjugating enzyme and its substrate has never been demonstrated in vivo. We first used the cross-linking assay to examine proximity between the regulated ERAD substrate Hmg2p and Ubc7p, the principal ubiquitin-conjugating enzyme employed in its degradation (3, 34). When Hmg2p was immunoprecipitated from cells incubated with increasing concentrations of the cross-linker DSP (56), Ubc7p coimmunoprecipitated with Hmg2p in a cross-linker concentration-dependent fashion (Fig. 1a). Another ER-associated ubiquitin-conjugating enzyme, Ubc6p, which does not significantly participate in the ubiquitination and degradation of Hmg2p (3, 34), did not cross-link to Hmg2p (Fig. 1a). In contrast to Hmg2p, Ubc7p did not demonstrate any significant cross-linking to Hmg1p (Fig. 1a), the exceedingly stable isozyme of HMGR (26, 31). Thus, only a ubiquitin-conjugating enzyme required for Hmg2p degradation cross-linked to Hmg2p, and this cross-linking was in large part restricted to degradation substrate and not a similar, stable protein.

View larger version (37K): [in this window] [in a new window]   FIG. 1.   Ubc7p cross-linked to degraded Hmg2p in a mevalonate pathway-regulated manner but not to stable Hmg1p. Interaction of 2HA-Ubc7p with 1myc-HMGR was assessed by the in vivo cross-linking assay. Cells were grown to mid-log phase and removed to amine-free medium. DSP was added at the indicated concentrations to separate aliquots of cells, and cross-linking proceeded at 30°C for 30 min. Lysates were prepared, and 1myc-Hmg2p was immunoprecipitated with antiserum raised against its catalytic domain. Immunoprecipitates were denatured under reducing conditions and analyzed by immunoblotting with either the 9E10 antibody (-myc) to detect 1myc-Hmg2p or the 12CA5 antibody (-HA) to detect 2HA-Ubc7p. (a) Ubc7p cross-linked to Hmg2p but did not cross-link to Hmg1p. Cross-linking assay was done with strains expressing either 1myc-Hmg2p or 1myc-Hmg1p and coexpressing either 2HA-Ubc7p or 3HA-Ubc6p. The reduced immunoreactivity of 1myc-HMGR with DSP at 400 µg/ml is due to modification of the lysine residue in the myc epitope sequence, not reduced immunoprecipitation of HMGR (unpublished observations). The small amount of 2HA-Ubc7p that is present with DSP at 0 µg/ml in this and the following figures is similar to that observed when preimmune serum is used instead of HMGR-specific antiserum (unpublished observations), indicating that it is nonspecific immunoprecipitation. (b) Ubc7p cross-linked to a degraded version of Hmg1p; cross-linking assay with strains expressing either 1myc-Hmg2p, 1myc-Hmg1p, or hydrophilic-1myc-Hmg1p and coexpressing 2HA-Ubc7p. (c) Addition of either lovastatin or L-659,699 decreased Ubc7p cross-linking. Regulated Ubc7p cross-linking to Hmg2p was observed by preincubating the cells with either no or 50 µg of lovastatin (Lov) or 10 µg of L-659,599 (L659) per ml for 2 h prior to addition of DSP. Cross-linking assay was performed as in Fig. 1. (d) Cross-linking of Ubc7p to 6myc-Hmg2p was not regulated by the mevalonate pathway. Ubc7p cross-linking to 6myc-Hmg2p was observed by preincubating the cells with either 0 or 50 µg lovastatin or 10 µg of L-659,599 per ml for 2 h prior to addition of DSP.

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In vivo action of the HRD ubiquitin ligase complex. Thus, Ubc7p cross-linking to ER proteins was directly correlated with their degradation status. Proteins that are naturally stable or are stabilized by physiological conditions within the cell did not cross-link to Ubc7p. Ubc7p-dependent degradation substrates did cross-link to Ubc7p, suggesting that Ubc7p directly associated with ERAD substrates in a highly specific and selective manner. In the case of regulated Hmg2p, the cross-linking between Hmg2p and Ubc7p was physiologically regulated in a manner entirely consistent with the regulation of Hmg2p ubiquitination and degradation by mevalonate pathway production.

HRD complex mediated Ubc7p cross-linking to substrates. We next investigated the role of the Hrd1p/Hrd3p ubiquitin ligase complex in promoting the proximity of Ubc7p to Hmg2p. To do so, we tested the effects of null alleles of the genes encoding these proteins on Hmg2p-Ubc7p cross-linking. As expected, when the individual null alleles were introduced into the strain that coexpressed the epitope-tagged versions of Hmg2p and Ubc7p, the presence of either the hrd1 or the hrd3 allele resulted in complete stabilization of Hmg2p through reduced ubiquitination (Fig. 2a and b, respectively). Consistent with their effect on Hmg2p ubiquitination and degradation, the presence of either the hrd1 or the hrd3 allele resulted in almost complete loss of Ubc7p cross-linking to Hmg2p (Fig. 2c). Importantly, the effects of the hrd1 and hrd3 alleles were not explained by altered steady-state levels or cellular distribution of Ubc7p. In both of these mutants, Ubc7p stability, steady-state levels, and microsomal association were all similar to those in wild-type cells (Fig. 2d and e). Similar to the hrd1 and hrd3 alleles, introduction of the cue1 allele, which results in loss of the Ubc7p ER membrane anchor Cue1p (5), also stabilized Hmg2p through reduced ubiquitination (Fig. 2a and b). The presence of the cue1 allele similarly reduced Ubc7p cross-linking to Hmg2p (Fig. 2c). However, loss of Cue1p prevented Ubc7p from associating with the ER membrane and resulted in rapid degradation of the subsequently soluble Ubc7p protein (Fig. 2d and e). Thus, reduced Ubc7p cross-linking to Hmg2p in cue1 cells was likely due to the mislocalization and reduced steady-state levels of Ubc7p. In contrast to the effects of the hrd1 and hrd3 alleles, introduction of the hrd2-1 allele, which stabilizes Hmg2p by altering proteasome activity but not Hmg2p ubiquitination (32) (Fig. 2a and b), had no effect on Ubc7p cross-linking to Hmg2p (Fig. 2c). From these combined results, the Hrd1p/Hrd3p complex was required to promote a functional proximity between Ubc7p and the target ERAD substrate Hmg2p, consistent with its proposed function as a RING-H2 ubiquitin ligase complex.

View larger version (52K): [in this window] [in a new window]   FIG. 2.   HRD gene-encoded proteins required for Ubc7p-Hmg2p cross-linking. (a) The appropriate null alleles of each gene required for Hmg2p degradation were introduced into the strain coexpressing 1myc-Hmg2p and 2HA-Ubc7p. Correct introduction of each null allele was determined by observation of 1myc-Hmg2p stabilization in a cycloheximide-chase assay, performed as in Fig. 5 (left panels). Lysates from each indicated time point after addition of cycloheximide were prepared and immunoblotted to determine the level of 1myc-Hmg2p. (b) The presence of cue1, hrd1, and hrd3 blocked Hmg2p-regulated ubiquitination. Ubiquitination assays of cells carrying the indicated null allele were performed in the presence of no drug or zaragozic acid (ZA, 10 µg/ml). Upper panels are the result of antiubiquitin (-Ub) immunoblotting for covalently linked Ub-Hmg2p conjugates. Lower panels are the result of parallel immunoblotting of an aliquot (1/8 total volume) of the same immunoprecipitates with the 9E10 anti-myc antibody (-myc) to assess total immunoprecipitated Hmg2p. (c) The appropriate null alleles of each gene required for Hmg2p degradation were introduced into the strain coexpressing 1myc-Hmg2p and 2HA-Ubc7p. Cross-linking assay was performed as in Fig. 1. (d) 2HA-Ubc7p levels and degradation were unaffected in hrd1 and hrd3 cells, but 2HA-Ubc7p was rapidly degraded in cue1 cells. Cells expressing 2HA-Ubc7p and the indicated hrd allele were grown to log phase. Lysates from each indicated time point after addition of cycloheximide were prepared and immunoblotted to determine the level of 2HA-Ubc7p. (e) Membrane fractionation of the cells from panel d was performed by osmotically lysing cells and preparing a crude microsomal fraction. The ability of 2HA-Ubc7p to remain membrane bound was assayed under conditions of buffer, 2.5 M NaCl, 2.5 M urea, 0.8 M potassium acetate (KOAc, pH 11.6), or 1% Triton X-100. Lanes S, supernatant fractions. Lanes P, pellet fractions.

Cross-linking of HRD ubiquitin ligase complex to substrates. The Hrd1p/Hrd3p ubiquitin ligase complex is required for the specific ubiquitination of ERAD substrates by Ubc7p and appeared to promote a functional association between Ubc7p and ERAD substrates. This suggested that the HRD complex itself associated with target substrates. Accordingly, we evaluated cross-linking of the HRD complex components Hrd1p and Hrd3p with ERAD substrates. Because Hmg2p degradation is regulated by the mevalonate pathway (25, 31), we were particularly interested in the role that potential HRD complex-substrate interactions played in substrate selection and physiological regulation.

View larger version (49K): [in this window] [in a new window]   FIG. 3.   Hrd1p/Hrd3p ubiquitin ligase complex cross-linked with both ERAD substrates and stable proteins. (a) Hrd1p cross-linked to both the degraded Hmg2p and the stable Hmg1p. Strains expressing either 1myc-Hmg2p or 1myc-Hmg1p and coexpressing 3HA-Hrd1p were subjected to the cross-linking assay as described for Fig. 1. 1myc-Hmg2p and 1myc-Hmg1p were detected using the 9E10 antibody (-myc), and 3HA-Hrd1p was detected using the 12CA5 antibody (-HA). The lower immunoreactivity of 1myc-HMGR with DSP at 400 µg/ml lane is due to modification of the lysine residue in the myc epitope sequence, not reduced immunoprecipitation of HMGR (data not shown). Preimmune serum substituted for anti-Hmg2p antiserum in the indicated sample is indicated as Pre. (b) Hrd3p cross-linked to both the degraded Hmg2p and the stable Hmg1p. Strains expressing either 1myc-Hmg2p or 1myc-Hmg1p and coexpressing 3HA-Hrd3p were subjected to the cross-linking assay as described for panel a. (c) Sec12p did not cross-link to Hmg2p. Strains expressing either 1myc-Hmg2p or 1myc-Hmg1p and coexpressing 3HA-Sec12p were subjected to the cross-linking assay. (d) Hrd1p cross-linking to Hmg2p was unregulated by the mevalonate pathway. Lack of regulated Hrd1p cross-linking to Hmg2p was observed by preincubating the cells with either no drug, lovastatin (Lov) (50 µg/ml), or L-659,599 (L659) (10 µg/ml) for 2 h prior to addition of DSP. (e) Hrd3p cross-linking to Hmg2p was unregulated by the mevalonate pathway. Lack of regulated Hrd3p cross-linking to Hmg2p was observed by preincubating the cells as in panel d.

View larger version (59K): [in this window] [in a new window]   FIG. 4.   Hrd1p/Hrd3p cross-linking to Hmg2p did not require the presence of Ubc7p or Cue1p. (a) Hrd3p cross-linking to Hmg2p in the presence of either the hrd1, ubc7, or cue1 allele. The appropriate null alleles of each gene required for Hmg2p degradation were introduced into the strain coexpressing 1myc-Hmg2p and 3HA-Hrd3p. Cross-linking assay was performed as in Fig. 1. (b) Hrd1p cross-linking to Hmg2p in the presence of either the hrd1, ubc7, or cue1 allele. The appropriate null alleles of each gene required for Hmg2p degradation were introduced into the strain coexpressing 1myc-Hmg2p and 3HA-Hrd1p, and the cross-linking assay was performed. (c) Hrd3p function was required for Hrd1p cross-linking to Hmg2p under normal Hrd1p expression levels. Cells expressing 1myc-Hmg2p and 3HA-Hrd1p from its native promoter and coexpressing either the wild-type HRD3 allele (wt), the hrd3 allele, or the truncated hrd3 allele (hrd3357-833) were subjected to the cross-linking assay. (d) Expression of Hrd3p357-833 as the only source of Hrd3p allowed stabilization of Hrd1p, but did not allow ERAD. Cycloheximide-chase assays of strains expressing 1myc-Hmg2p and either 3HA-Hrd1p or 2HA-Ubc7p and containing either the wild-type HRD3 allele, the hrd3 allele, or the truncated hrd3 allele. Cells were grown to mid-log phase (OD600 of 0.5), and cycloheximide was added to a final concentration of 50 µg/ml. Lysates were prepared at the indicated time points. The 9E10 anti-myc antibody was used to detect 1myc-Hmg2p. An anti-HA ascites fluid was used to detect 3HA-Hrd1p and 2HA-Ubc7p. (e) Hrd3p function was required for Ubc7p cross-linking to Hmg2p under normal Hrd1p expression levels. Cells expressing 1myc-Hmg2p, 2HA-Ubc7p, and Hrd1p from its native promoter and coexpressing either the wild-type HRD3 allele, the hrd3 allele, or the truncated hrd3 allele, were subjected to the cross-linking assay as in Fig. 1. (f) The N-terminal region of Hrd3p was required for Hrd3p cross-linking to Hmg2p. Cells expressing 1myc-Hmg2p and carrying the wild-type HRD3 allele, the hrd3 allele, or the hrd3357-833 allele were subjected to the cross-linking assay as in Fig. 3.

Hmg2p is recognized for degradation by a regulated structural change. Both Hrd1p and Hrd3p in the HRD ubiquitin ligase complex appeared to associate indiscriminately with both stable proteins and ERAD substrates. However, the complex specifically mediated functional Ubc7p cross-linking with proteins slated for either constitutive or regulated ERAD. Thus, the Hrd1p/Hrd3p ubiquitin ligase complex discriminated ERAD substrates from stable proteins at the level of Ubc7p recruitment through some feature of the targeted protein that distinguishes it as an ERAD substrate. Considering the quality control function of the HRD complex, a reasonable criterion for ERAD substrates would be molecular hallmarks consistent with misfolded or incompletely folded proteins.

View larger version (33K): [in this window] [in a new window]   FIG. 5.   Chemical chaperone glycerol increased the steady-state level of Hmg2p and blocked the degradation-enhancing effect of zaragozic acid. (a) Incubation of cells with glycerol increased the steady-state levels of Hmg2p-GFP. Cells expressing the indicated HMGR-GFP variant were grown in minimal medium to mid-log phase in the presence or absence of glycerol (final concentration, 10%). Lovastatin (final concentration, 25 µg/ml) was added to the indicated sample, and all samples were incubated at 30°C for an additional 4 h. Steady-state GFP fluorescence was analyzed by flow cytometry. (b) Glycerol did not affect mevalonate pathway production. TLC analysis of the nonsaponifiable lipid fraction from yeast cells treated with no drug (nd), 10% glycerol (G), or 50 µg of lovastatin (L) per ml. Cells were incubated with [14C]acetate in the presence of the indicated drug for 6 h. Lipids were extracted and gently saponified, and the nonsaponifiable lipid fraction was extracted and applied to a TLC plate. Migration of standards is marked on the right. (c) Glycerol blunted the degradation-enhancing effect of zaragozic acid. Cells expressing Hmg2p-GFP were grown in minimal medium to mid-log phase. Zaragozic acid (ZA; final concentration, 10 µg/ml) and/or glycerol (final concentration, 10%) was added to the indicated samples, which were incubated at 30°C for an additional 4 h. Steady-state GFP fluorescence was analyzed by flow cytometry.

View larger version (37K): [in this window] [in a new window]   FIG. 6.   Chemical chaperone glycerol stabilized Hmg2p and reduced Ubc7p cross-linking to Hmg2p. (a) Glycerol stabilized 1myc-Hmg2p but not 6myc-Hmg2p. Cells expressing either 1myc-Hmg2p or 6myc-Hmg2p were grown in minimal medium to mid-log phase. Glycerol (final concentration, 15%) or lovastatin (lovastatin; final concentration, 25 µg/ml) was added to the indicated samples, and the cells were incubated at 30°C for 4 h. Cells were then subjected to a cycloheximide-chase assay. Lysates were prepared at the indicated time points after cycloheximide addition and immunoblotted with the 9E10 antibody to assess the levels of myc-HMGR. (b and c) Glycerol blocked Ubc7p cross-linking to Hmg2p but not 6myc-Hmg2p. Cells coexpressing either 1myc-Hmg2p or 6myc-Hmg2p and 2HA-Ubc7p were grown in minimal medium to mid-log phase. Cells were treated with either no drug, 15% glycerol, or lovastatin (25 mg/ml) for 2 h at 30°C. A similar cross-linking assay was performed as in Fig. 1. (d) Glycerol did not block Hrd1p cross-linking to Hmg2p. Cells coexpressing 1myc-Hmg2p and 3HA-Hrd1p were grown in minimal media to mid-log phase. Cells were treated with either no drug or 15% glycerol for 2 h at 30°C. A similar cross-linking assay was performed as in Fig. 4.

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View larger version (70K): [in this window] [in a new window]   FIG. 7.   Hmg2p transmembrane domain underwent structural transition from stable to degradation-competent state. Limited proteolytic analysis of intact microsomes containing 1myc-Hmg2p revealed an altered sensitivity to trypsin when cells were incubated with zaragozic acid. Cells expressing 1myc-Hmg2p were grown to mid-log phase in minimal medium. The cells were treated with either no drug (nd), lovastatin (L; 100 µg/ml, final concentration) for 30 min, zaragozic acid (Z; 10 µg/ml, final concentration) for 15 min, or both lovastatin and zaragozic acid (ZL) for 30 min at 30°C. These treatments did not appreciably alter the steady-state level of Hmg2p during the period of treatment (unpublished observations). Intact, isolated microsomes were prepared from these cells. Separate aliquots of isolated microsomes were treated with trypsin (0.5 µg/ml) for 30 min at 0°C in the absence of detergent. The degree of protease digestion was observed by immunoblotting with a polyclonal antiserum specific for Hmg2p sequences located in both the lumen and cytosol. All samples had similar amounts of undigested Hmg2p in the microsomes prior to treatment with trypsin (no-trypsin lanes, left panel). All total-protein loads were identical in each lane, as assessed from India ink staining of the Western blot (data not shown). Full-length Hmg2p is indicated with an arrow. Lower-molecular-weight bands are indicated with the brackets. The asterisk indicates a nonspecific band that cross-reacts with the anti-Hmg2p antiserum.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

The ER-associated proteins Hrd1p and Hrd3p function as a RING-H2 ubiquitin ligase complex required for the targeting and ubiquitination of ER-associated, proteasome-dependent degradation substrates (3, 27), such as the ER lumenal protein CPY* (6, 67) and the ER membrane protein Hmg2p (27, 32). A current model for RING-H2 ubiquitin ligase action is that the ubiquitin ligase mediates direct association between the substrate and the appropriate ubiquitin-conjugating enzyme, allowing the specific transfer of ubiquitin to the target protein. From our studies, we demonstrate that the HRD complex does mediate functional association between the ER-associated ubiquitin-conjugating enzyme Ubc7p and ERAD substrates through a unique substrate-targeting mechanism, which may act in an inspection process to target ERAD substrates for ubiquitination and subsequent proteasome-dependent degradation.

Mechanism of substrate selection: a scanning mechanism for the HRD complex. Ubiquitin ligases must interact with substrates as part of the targeting mechanism. In the cases of other characterized ubiquitin ligases, specific sequence motifs in the substrate serve as key specificity determinants for ubiquitin ligase binding and subsequent ubiquitination (1, 2, 19, 23, 41, 42, 47, 53, 54, 57, 62, 63, 76, 77, 90). Unlike other ubiquitin ligase proteins and associated components, however, both Hrd1p and Hrd3p associated with stable proteins in addition to degradation substrates (Fig. 8). Thus, we suggest that the Hrd1p/Hrd3p complex "scans" the ER for candidate substrates, querying all accessible proteins and associating with stable proteins and degradation substrates alike. When a bound protein meets the criteria for ERAD, the HRD complex mediates a functional association between the substrate and the ER-associated ubiquitin conjugating enzyme Ubc7p, thereby allowing direct transfer of ubiquitin from Ubc7p to the substrate (Fig. 8). Substrate specificity lies not in substrate-HRD complex association, but in the subsequent HRD-dependent functional association of the ubiquitin-conjugating enzyme with the substrate. A reasonable criterion for the HRD complex to proceed with functional association of Ubc7p with a substrate to allow substrate ubiquitination would be recognition of hallmarks of misfolding. By this model, the difference between a degradation substrate and a stable protein would be the exposure of such hallmarks by the degradation substrate but not the stable protein. In this regard, the soluble Hrd1p C-terminal domain, which contains the RING-H2 motif, does preferentially program the ubiquitination of a misfolded protein in an in vitro ubiquitination assay (3), indicating that Hrd1p might participate directly in some aspect of quality control recognition.

View larger version (40K): [in this window] [in a new window]   FIG. 8.   Model for HRD ubiquitin ligase complex association with Hmg2p and mechanism of mevalonate pathway-regulated ERAD. (a) Stable proteins associate with Hrd1p/Hrd3p core complex, but the structure of the stable protein does not allow functional association with Ubc7p. Hmg2p stabilized as a result of low mevalonate pathway production would have similar interaction kinetics with the HRD ubiquitin ligase complex. (b) ERAD substrates associate with Hrd1p/Hrd3p core complex, and their misfolded or mutant structures allow functional association with Ubc7p mediated by the Hrd1p/Hrd3p complex. A structural change within Hmg2p as a result of abundant production of downstream molecules in the mevalonate pathway would result in Hmg2p's acquisition of a similar "misfolded" state as other ERAD substrates.

Mechanism(s) of Ubc7p recruitment. The fact that Ubc7p cross-linked only to degradation substrates and not to stable proteins leads to a few possible models for Ubc7p recruitment to the substrate by the Hrd1p/Hrd3p complex. First, Ubc7p may not be constitutively associated with the Hrd1p/Hrd3p core complex and may only be recruited to join the complex when a degradation substrate is bound by Hrd1p and Hrd3p, possibly as the result of an allosteric alteration in the Hrd1p/Hrd3p complex induced only by degradation substrate binding. Alternatively, Ubc7p may be constitutively associated with the core Hrd1p/Hrd3p complex, but is not in the correct position to effect ubiquitination of a bound protein. Again, only upon the binding of a degradation substrate by the Hrd1p and Hrd3p would the complex undergo an allosteric alteration that subsequently brings Ubc7p into a correct orientation with the substrate to effect substrate ubiquitination. Lastly, Ubc7p may be constitutively associated with the Hrd1p/Hrd3p core complex in a fixed position conducive to effect substrate ubiquitination so that the complex need not undergo any structural alteration. Instead, the complex distinguishes degradation substrates from stable proteins by the misfolded structure of the degradation substrate that allows Ubc7p direct access to the bound protein's lysine residues, which are used as the sites for the covalent attachment of ubiquitin (21, 46). Only those proteins to be ubiquitinated and degraded would have lysine residues accessible to Ubc7p.

Role of each component of the complex in substrate binding. Consistent with its role as a ubiquitin ligase (3), Hrd1p was required for Ubc7p cross-linking to ERAD substrates. The cytosolic RING-H2 domain of Hrd1p mediates direct recruitment of Ubc7p, as this domain is required for direct, in vivo and in vitro interaction between Hrd1p and Ubc7p (3, 16). The RING-H2 motif similarly binds ubiquitin-conjugating enzymes in other homologous ubiquitin ligases (47, 57), and the crystal structure between cCbl and UbcH7 demonstrated that the cCbl RING-H2 motif mediates direct interaction between the ubiquitin ligase and the ubiquitin-conjugating enzyme (92). Hrd1p directly binds Ubc7p (3, 16) and directs Ubc7p proximity to substrates, so it is likely that Hrd1p contains some substrate-binding activity. Because Hrd1p directs ubiquitination of only ERAD substrates, it must have a way of distinguishing between the degradation substrates and the stable proteins that it interacts with. It is possible that Hrd1p may engage all proteins with its transmembrane domain but only binds ERAD substrates with its RING-H2 domain, thus bringing Ubc7p in proximity to the degradation substrate. In fact, the cytosolic domain of Hrd1p has a preference for misfolded proteins in an in vitro ubiquitination assay (3), possibly indicating that the Hrd1p cytosolic domain possesses a component of substrate recognition. Hrd1p functions in in vivo ERAD in the absence of Hrd3p when expressed at sufficiently high levels (27, 67), further supporting the idea of a substrate recognition component within Hrd1p.

Implications of Hmg2p interaction with the HRD complex. The structural transition mechanism for Hmg2p-regulated degradation has several important implications. It has recently been demonstrated that regulated degradation of mammalian HMGR is mediated by ubiquitination (69), in a manner very similar to Hmg2p (34). Although some differences exist in the molecular details of the two systems, it is very likely that the same mechanism of signal-induced transition to a quality control substrate underlies this important axis of sterol synthesis regulation. At present, it is not clear if the ability to undergo this structural transition is an autonomous feature of the HMGR molecule or if it is mediated by ancillary machinery that brings the transition about. In either case, however, it is possible that small molecules could be designed or discovered to drive the transition in a specific and clinically useful manner. More generally, as quality control degradation systems likely exist in the cytosol, and quite possibly the nucleus or mitochondria, it may be that entry of substrates into these systems occurs by similar mechanisms and could be harnessed in much the same fashion as the ER quality control degradation apparatus. The observation of a regulated transition to a quality control substrate allows the framing of both basic and clinical lines of inquiry to examine the utility and biological generality of this mode of protein regulation.