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Molecular and Cellular Biology, July 2001, p. 4276-4291, Vol. 21, No. 13
Section of Cell and Developmental Biology,
Division of Biology, University of California, San Diego, La Jolla,
California 92093
Received 18 December 2000/Returned for modification 6 February
2001/Accepted 23 March 2001
Ubiquitination is used to target both normal proteins for specific
regulated degradation and misfolded proteins for purposes of quality
control destruction. Ubiquitin ligases, or E3 proteins, promote
ubiquitination by effecting the specific transfer of ubiquitin from the
correct ubiquitin-conjugating enzyme, or E2 protein, to the target
substrate. Substrate specificity is usually determined by specific
sequence determinants, or degrons, in the target substrate that are
recognized by the ubiquitin ligase. In quality control, however, a
potentially vast collection of proteins with characteristic hallmarks
of misfolding or misassembly are targeted with high specificity despite
the lack of any sequence similarity between substrates. In order to
understand the mechanisms of quality control ubiquitination, we have
focused our attention on the first characterized quality control
ubiquitin ligase, the HRD complex, which is responsible for
the endoplasmic reticulum (ER)-associated degradation (ERAD) of
numerous ER-resident proteins. Using an in vivo cross-linking assay, we
directly examined the association of the separate HRD complex components with various ERAD substrates. We have discovered that the HRD ubiquitin ligase complex associates with both
ERAD substrates and stable proteins, but only mediates
ubiquitin-conjugating enzyme association with ERAD substrates. Our
studies with the sterol pathway-regulated ERAD substrate Hmg2p, an
isozyme of the yeast cholesterol biosynthetic enzyme HMG-coenzyme A
reductase (HMGR), indicated that the HRD complex discerns
between a degradation-competent "misfolded" state and a stable,
tightly folded state. Thus, it appears that the physiologically
regulated, HRD-dependent degradation of HMGR is effected by
a programmed structural transition from a stable protein to a quality
control substrate.
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 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.
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.13.4276-4291.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
In Vivo Action of the HRD Ubiquitin
Ligase Complex: Mechanisms of Endoplasmic Reticulum Quality Control and
Sterol Regulation
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
::LEU2
was constructed as follows. A 3.1-kb XhoI-SpeI
fragment from pRH508 which contained the HRD3 gene
(32) was inserted between the BamHI and
EcoRI sites in pBluescript KSII, resulting in pRH1175. The
LEU2 gene was PCR amplified from pRS405, digested with
XbaI, and inserted between the BsaBI and
NheI sites in pRH1175. The resulting
hrd3::LEU2 plasmid was named pRH1185.
The plasmid used to make ubc7::LEU2
was constructed as follows. A 650-bp fragment containing the coding
region for the UBC7 gene was PCR amplified from pRH685 and
inserted between the PstI and SalI sites in
pBluescript KSII, which resulted in pRH1176. pRH1176 contained an
HpaI and a BsrGI site with the UBC7
sequence. A 1.5-kb HpaI-BsrGI fragment from
pRS405 containing the LEU2 gene was inserted between the
HpaI and BsrGI sites in pRH1176. The resulting
ubc7::LEU2 plasmid was named pRH1186.
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).
his3200 lys2-801 ade2-101
leu2 ura3-52::1MYC-HMG2::URA3
trp1::HISG met2 hmg1::LYS2
HMG2) and RHY1915 (MAT his3200 lys2-801 ade2-101
leu2
ura3-52::1MYC-HMG1::URA3 trp1::HISG met2 hmg1::LYS2
HMG2) were used as parent strains. The plasmid containing
3HA-Hrd1p, pRH1196, was digested with BglII and integrated
at the HRD1 genomic locus. The plasmid containing 3HA-Hrd3p,
pRH1263, was digested with BsrGI and integrated at the
HRD3 genomic locus. The plasmid containing 2HA-Ubc7p,
pRH373, was digested with BsgI and integrated at the
TRP1 genomic locus. The plasmid containing 3HA-Ubc6p,
pRH1217, was digested with BsrGI and integrated at the
UBC6 genomic locus. Transformants were selected for
TRP+ prototrophy. The plasmid containing
hrd1::LEU2, pRH1184
(85), was digested with XhoI and
BamHI and integrated at the HRD1 genomic locus.
The plasmid containing hrd3::LEU2,
pRH1185, was digested with XhoI and NdeI and
integrated at the HRD3 genomic locus. The plasmid containing
ubc7::LEU2, pRH1186, was digested
with PstI and SalI and integrated at the
UBC7 genomic locus. The plasmid containing
cue1::LEU2, pTX118 (5),
was digested with ApaI and SacI and integrated at
the CUE1 genomic locus. Transformants were selected for
LEU+ prototrophy.
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.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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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.
|
63-219 (Fig.
1d), a grossly misfolded, epitope-tagged mutant of Hmg2p missing 157 residues of the native Hmg2p sequence, including a putative
transmembrane span that undergoes constitutive, unregulated Ubc7p-dependent degradation (25, 32).
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.
|
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.
To examine such interactions, we used strains that coexpressed either 3HA-Hrd1p or 3HA-Hrd3p and a relevant 1myc-HMGR protein. Using the same in vivo cross-linking assay, we observed that both Hrd1p and Hrd3p cross-linked to Hmg2p (Fig. 3a and b, respectively). Surprisingly, and unlike Ubc7p cross-linking, both Hrd1p and Hrd3p also cross-linked to the stable HMGR isozyme Hmg1p (Fig. 3a and b, respectively). Furthermore, and consistent with this observation, cross-linking of either Hrd1p or Hrd3p to Hmg2p was completely unaffected by incubation of cells with lovastatin or L-659,699 (Fig. 3d and e, respectively), which stabilized Hmg2p (31, 34) and blocked Ubc7p cross-linking to Hmg2p (Fig. 1c). Thus, the Hrd1p/Hrd3p complex appeared to associate with both stable and degraded proteins alike and association with Hmg2p was not regulated. The cross-linking of Hrd1p and Hrd3p to both Hmg1p and Hmg2p did not appear to be due simply to nonspecific cross-linking of all ER proteins to Hmg1p or Hmg2p, as other ER proteins not involved in HRD-dependent degradation did not cross-link to either Hmg1p or Hmg2p, such as Ubc6p (Fig. 1a) and Sec12p (Fig. 3c).
|
,
ubc7, or cue1 allele (Fig.
4a and unpublished observations),
indicating that Hrd3p association with ER proteins was independent of
Hrd1p, Ubc7p, or Cue1p expression. Hrd1p cross-linking to Hmg2p or
Hmg1p was equivalent in wild-type cells and cells carrying the
ubc7 or cue1 allele, but was strongly
diminished by the presence of the hrd3 allele (Fig. 4b
and unpublished observations). However, Hrd1p steady-state levels are
eightfold lower in hrd3 cells due to its rapid
degradation (27) (Fig. 4d).
|
allele.
Furthermore, loss of the N-terminal region also resulted in greatly
reduced Hrd3p cross-linking with substrate (Fig. 4f). Thus, the Hrd3p
N-terminal region was required for Hrd3p-substrate association and
appeared to be critical in controlling Hrd1p-Ubc7p cross-linking with
substrate under normal Hrd1p steady-state levels. Accordingly, Hrd3p
functions both in the maintenance of Hrd1p levels and in a separate
ERAD function required to target Ubc7p and Hrd1p to the substrate.
Importantly, functional disruption of Hrd1p and Hrd3p cross-linking to
ERAD substrates indicated that both Hrd1p and Hrd3p cross-linking was physiologically relevant.
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.
According to this model, constitutively degraded substrates would always cross-link with Ubc7p, but physiologically regulated substrates would only cross-link with Ubc7p when degradation signals were high. In fact, both the Hmg2p degradation rate and HRD-dependent Ubc7p cross-linking to Hmg2p are altered accordingly by a feedback regulatory signal generated from the conditions of cellular mevalonate pathway production (25, 31) (Fig. 1c). Because the HRD complex targets misfolded proteins for degradation, we investigated if a mevalonate pathway-regulated alteration in the Hmg2p folding state programmed its controlled ERAD by the quality control HRD complex. We used a class of compounds known as chemical chaperones to examine the role of the Hmg2p folding state in HRD complex recognition, Ubc7p interaction, and subsequent degradation of Hmg2p. Such agents have been demonstrated to enhance the folding of various mutant and normal proteins in cell and whole-animal studies (8, 9, 73). Importantly, addition of such chemical chaperones to cells has been shown to block substrate-specific ERAD by enhancing the folding of a number of mutant, misfolded proteins. For example, addition of the chemical chaperone glycerol to mammalian cells enhances the folding of the cystic fibrosis transmembrane conductance regulator (CFTR)F508 mutant, which is normally retained in the ER and
destroyed by ERAD (82), thereby allowing passage of the mutant CFTR through the secretory pathway, restoring normal
CFTR-mediated chloride ion transport into the cell (8,
73). Similarly, addition of the chemical chaperone
4-phenylbutyric acid to mammalian cells enhances folding and secretion
of the PiZ isoform of 1-antitrypsin (10), which is also
normally retained in the ER and subject to ERAD (13).
We first determined if addition of the chemical chaperone glycerol to
yeast cells could stabilize the normally regulated fluorescence reporter protein Hmg2p-GFP (15, 25, 33). When cells
expressing Hmg2p-GFP were grown in 10% glycerol, the Hmg2p-GFP
steady-state level was significantly elevated, which was observed as a
rightward shift in the fluorescence histogram of glycerol-treated cells compared to untreated cells (Fig. 5a).
Such an increase in Hmg2p-GFP steady-state level is normally indicative
of Hmg2p-GFP stabilization (15, 25, 26, 33). In contrast,
identical treatment of a strain expressing the extremely stable
Hmg1p-GFP resulted in only a minor alteration in cellular fluorescence
in either case (Fig. 6a). The increase in
Hmg2p-GFP steady-state levels in glycerol-treated cells was identical
to that in cells treated with lovastatin (Fig. 5a). Importantly,
glycerol did not alter production of downstream mevalonate pathway
products (Fig. 5b). Furthermore, addition of lovastatin to
glycerol-treated cells had no added effect on the glycerol-induced
increase in Hmg2p-GFP steady-state levels (unpublished observations).
Not only did incubation of cells with glycerol increase Hmg2p-GFP
steady-state levels, but the normal, zaragozic acid-induced reduction
in Hmg2p-GFP steady-state levels was also completely blocked by
addition of glycerol at the same time as zaragozic acid (Fig. 5c).
Zaragozic acid is an inhibitor of the downstream enzyme squalene
synthase and results in enhanced Hmg2p ubiquitination and degradation
through build-up of the pathway molecule farnesyl diphosphate
(25, 34).
|
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63-219, a grossly misfolded mutant of Hmg2p
that is missing 157 residues of the native Hmg2p sequence
(32), including a putative transmembrane span. This large
deletion of Hmg2p sequence would prevent
6myc-Hmg2p63-219 from ever achieving a normally folded
sequence, and consistent with this, addition of glycerol did not
significantly affect the steady-state level of
6myc-Hmg2p63-219 (Fig. 5a, right panel).
It was likely that glycerol's effect occurred through stabilization of
Hmg2p, possibly by enhancing Hmg2p folding. Therefore, we directly
determined the effect of glycerol on the degradation of regulated
1myc-Hmg2p and grossly misfolded 6myc-Hmg2p63-219 by
the cycloheximide-chase assay. Incubation of cells in glycerol resulted
in stabilization of 1myc-Hmg2p to a similar extent as incubation of
cells with lovastatin (Fig. 6a). In a similar experiment, the
constitutive degradation of 6myc-Hmg2p63-219 was unaltered by incubation of cells with glycerol (Fig. 6a), consistent with its severely perturbed structure (26, 32).
Finally, we examined the effect of glycerol in the cross-linking
between Ubc7p and Hmg2p. When cells were incubated with glycerol, Ubc7p
cross-linking to Hmg2p was reduced, and the effect was similar to
incubation of cells with lovastatin (Fig. 6b). This was not the result
of decreased cross-linking efficiency for Ubc7p, as Ubc7p cross-linked
similarly to 6myc-Hmg2p63-219 in both the presence and
absence of glycerol (Fig. 6c). This was also not a result of decreased
Ubc7p stability or membrane association (unpublished observations), nor
was it a result of decreased Hrd1p cross-linking to substrate, as Hrd1p
cross-linked to Hmg2p identically in the presence and absence of
glycerol (Fig. 6d). Thus, enhanced folding of Hmg2p specifically
blocked functional HRD-mediated proximity between Ubc7p and
Hmg2p and was without effect on HRD complex association with substrate.
These results supported a model in which the HRD complex
detects proteins with appropriate structural features and promotes functional proximity between Ubc7p and the substrate to initiate ERAD.
The glycerol experiments implied that the Hmg2p structure was different
between the stable state and the degradation-competent state, with the
stable state having a more completely folded structure. To determine if
Hmg2p did have a dynamic structure, we examined the sensitivity of the
Hmg2p structure to exogenously added proteases under different
mevalonate pathway conditions. A similar assay has been employed
successfully to evaluate the structural differences between the
wild-type form of CFTR and the rapidly degraded CFTR F508 mutant
(91). We probed the protease sensitivity of Hmg2p in
isolated microsomes from yeast cells treated with either no drug,
stabilizing lovastatin, or the degradation-enhancing zaragozic acid.
A noticeable and reproducible difference was observed in the
proteolytic sensitivity of Hmg2p from the zaragozic acid-treated cells
compared to the no-drug cells and the lovastatin-treated cells (Fig.
7, right panels, lanes Z versus nd and
L). The complete absence of lower-molecular-weight fragments in the
zaragozic acid-treated cells suggested that Hmg2p from zaragozic
acid-treated cells had a different structure that altered its
sensitivity to exogenously added proteases. Furthermore, the reduced
presence of these fragments in the untreated sample correlated with its
intermediate rate of degradation. The change in Hmg2p proteolytic
sensitivity seen with the zaragozic acid-treated cells was blocked by
incubation of these cells in lovastatin prior to addition of zaragozic
acid (Fig. 7, right panels, lane ZL), indicating that the effect was regulated by upstream blockade of the mevalonate pathway. Thus, in a
manner consistent with the mevalonate pathway's regulation of Hmg2p
ubiquitination and degradation, Hmg2p had a differential sensitivity to
exogenously added proteases that depended on production of downstream
molecules of the mevalonate pathway. These results, and those with the
chemical chaperone, strongly suggest that Hmg2p undergoes a mevalonate
pathway-regulated structural transition that allows it to be recognized
by the HRD complex as a degradation substrate.
|
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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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.
|
a regulated ERAD
substrate, a constitutive ERAD substrate, and a stable protein not
involved in ER quality controlis not available for lumenal proteins.
A well-characterized, constitutively degraded, lumenal ERAD substrate
does exist in CPY* (6, 20, 37, 67), and we are initiating
similar studies to determine CPY*-HRD complex interactions.
Without a regulated lumenal ERAD substrate and a stable lumenal protein
not involved in ER quality control, it will be difficult to extend the
studies that we have reported here.
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.
In this regard, our studies delineating the sequence determinants required for Hmg2p-regulated degradation may be instructive. HRD-dependent ubiquitination of Hmg2p requires two distinct lysine residues, Lys6 and Lys357, which appear to be specifically involved in regulation of Hmg2p stability and may serve as the ubiquitination sites within Hmg2p (26). Perhaps the regulated Hmg2p structural transition brings about repositioning of Lys6 and Lys357, allowing for their close proximity to Ubc7p when Hmg2p is engaged by the HRD complex, which would result in their subsequent ubiquitination. Alternatively, Lys6 and Lys357 may act directly to promote global structural transitions of Hmg2p in response to signals from the mevalonate pathway. In this case, Lys6 and Lys357 would not be the recipients of Ubc7p-dependent ubiquitination, but together would be intimately involved in the structural changes that bring other lysine residues in close proximity to Ubc7p when Hmg2p is engaged by the HRD complex. Resolving the details of the regulated Hmg2p structural transition and the general ERAD scanning mechanism is a central concern of our ongoing work and may provide the key to understanding the mode of HRD complex-dependent Ubc7p recruitment to the substrate. In addition to Ubc7p, Ubc1p has recently been shown to be directed by Hrd1p in vivo to program the ubiquitination of both CPY* and Hmg2p (3, 22). This suggests that the HRD complex also mediates an interaction between ERAD substrates and Ubc1p, although this has yet to be tested.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.
To interact with such a diverse array of proteins, the complex must have a general binding mechanism. We cannot rule out that the complex recognizes specific sequences in each target degradation substrate, such as degrons (81), but no consensus sequence has been revealed through our mutagenic or computer analyses (26; unpublished observations). In fact, our recent analysis of the Hmg2p sequence (26) and our studies here indicate that the complex recognizes structural cues within Hmg2p rather than specific sequences. To allow recognition of common structural features among a variety of proteins, perhaps a protein in the complex has a substrate-binding site with binding affinities similar to those of a chaperone. It is quite possible that the initial substrate-scanning site is in the lumen of the ER, where both lumenal proteins and ER membrane proteins would exist. Hrd3p is the only protein of the established ER ubiquitin ligase complex that is almost entirely lumenal (27, 67, 71) and, from our cross-linking data, the only protein that interacted with all tested substrates in vivo in the absence of the other complex components when present at normal steady-state levels. These observations suggest that Hrd3p may serve a primary role in initial substrate recognition. Hrd3p may also function directly to control Hrd1p RING-H2 ubiquitin ligase activity (27), which promotes proximity between Ubc7p and the substrate, thereby indirectly controlling Ubc7p ubiquitin-conjugating activity. In support of this, a completely lumenal version of Hrd3p functions normally in ERAD (27), indicating that the action of Hrd3p to program the cytosolic ubiquitination of substrates occurs through a lumen-to-cytosol signaling process. Because Hrd3p interacts directly with the Hrd1p transmembrane domain (27), Hrd3p action to allow cytosolic ERAD events is likely transduced through the Hrd1p transmembrane domain. Thus, a defunct signaling process, through either elimination of Hrd1p or loss of Hrd3p function, may prevent Ubc7p association with substrates. In fact, elimination of Hrd1p did prevent Ubc7p cross-linking to substrates. Loss of Hrd3p function, by expression of only the Hrd3p N-terminal truncation mutant, also prevented Ubc7p cross-linking to substrates despite maintenance of normal Hrd1p levels (27). Additionally, although Hrd1p was maintained at normal levels by the Hrd3p truncation mutant, the Hrd3p truncation mutant did not allow Hrd1p cross-linking to substrates. This was most likely the result of an inability of the Hrd3p truncation mutant itself to associate with substrates and is consistent with the Hrd3p N-terminal region's functioning in the initial substrate recognition of the complex. Perhaps the initial binding of substrate with Hrd3p induces allosteric changes in the complex that result in the activation of Hrd1p ubiquitin ligase function. These allosteric changes would initiate correct temporal and spatial activation of Hrd1p RING-H2 ubiquitin ligase function and would promote physical association between ERAD substrates and Hrd1p and Ubc7p, leading to ubiquitination of the bound substrate. It may be that the truncated Hrd3p maintains Hrd1p in an inactive conformation, preventing any functional association of Hrd1p and Ubc7p with substrate. However, the deletion of such a large portion of Hrd3p may result in an aberrant structure of the remaining part of Hrd3p, possibly confounding this interpretation. The action of the HRD ubiquitin ligase complex involves coordination of processes and information on both sides of the ER membrane (27). One likely model for this coordination is that degradation proceeds by interaction of the Hrd1p/Hrd3p complex with a substrate engaged by the Sec61p retrotranslocation complex, which is required for ER degradation (94). The combined interaction of both Hrd3p and Hrd1p with the Sec61p-engaged substrate would allow the Hrd1p RING-H2 domain to promote Ubc7p association with substrate, resulting in subsequent ubiquitination of the substrate. Further exploration of the mechanism by which the HRD ubiquitin ligase complex operates on both sides of a membrane will be important in understanding specific aspects of ERAD and processes that employ ubiquitination as a mode of transmembrane signal transduction.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.
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ACKNOWLEDGMENTS |
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We thank Merck for the generous gifts of zaragozic acid, L-659,699, and lovastatin. R.G.G. thanks C. Melissa Morelli for essential technical support required to achieve strong signal intensity despite limited application of reagents. R.Y.H. thanks J. Theriot for opening unexpected conceptual doors.
This work was supported by NIH grant DK5199601 (R.Y.H.) and a Searle Scholarship (R.Y.H.).
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FOOTNOTES |
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* Corresponding author. Mailing address: Section of Cell and Developmental Biology, Division of Biology, University of California, San Diego, La Jolla, CA 92093. Phone: (858) 822-0511. Fax: (858) 534-0555. E-mail: rhampton{at}biomail.ucsd.edu.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
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Molecular and Cellular Biology, July 2001, p. 4276-4291, Vol. 21, No. 13
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.13.4276-4291.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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