From the Cellular and Molecular Biology Program and
Department of Horticulture, University of Wisconsin-Madison, Madison,
Wisconsin 53706 and § Department of Cell Biology, Harvard
Medical School, Boston, Massachusetts 02115
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ABSTRACT |
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The 26 S proteasome is a multisubunit proteolytic
complex responsible for degrading eukaryotic proteins targeted by
ubiquitin modification. Substrate recognition by the complex is
presumed to be mediated by one or more common receptor(s) with affinity for multiubiquitin chains, especially those internally linked through
lysine 48. We have identified previously a candidate for one such
receptor from diverse species, designated here as Mcb1 for
Multiubiquitin chain-binding
protein, based on its ability to bind Lys48-linked
multiubiquitin chains and its location within the 26 S proteasome
complex. Even though Mcb1 is likely not the only receptor in yeast, it
is necessary for conferring resistance to amino acid analogs and for
degrading a subset of ubiquitin pathway substrates such as
ubiquitin-Pro--galactosidase (Ub-Pro-
-gal) (van Nocker, S.,
Sadis, S., Rubin, D. M., Glickman, M., Fu, H., Coux, O., Wefes, I., Finley, D., and Vierstra, R. D. (1996) Mol. Cell.
Biol. 16, 6020-28). To further define the role of Mcb1 in
substrate recognition by the 26 S proteasome, a structure/function
analysis of various deletion and site-directed mutants of yeast and
Arabidopsis Mcb1 was performed. From these studies, we
identified a single stretch of conserved hydrophobic amino acids
(LAM/LALRL/V (ScMcb1 228-234 and AtMcb1 226-232)) within the
C-terminal half of each polypeptide that is necessary for interaction
with Lys48-linked multiubiquitin chains. Unexpectedly, this
domain was not essential for either Ub-Pro-
-gal degradation or
conferring resistance to amino acid analogs. The domain responsible for
these two activities was mapped to a conserved region near the N
terminus. Yeast and Arabidopsis Mcb1 derivatives containing
an intact multiubiquitin-binding site but missing the N-terminal region
failed to promote Ub-Pro-
-gal degradation and even accentuated the
sensitivity of the yeast
mcb1 strain to amino acid
analogs. This hypersensitivity was not caused by a gross defect in 26 S
proteasome assembly as mutants missing either the N-terminal domain or
the multiubiquitin chain-binding site could still associate with 26 S
proteasome and generate a complex indistinguishable in size from that
present in wild-type yeast. Together, these data indicate that residues
near the N terminus, and not the multiubiquitin chain-binding site, are
most critical for Mcb1 function in vivo.
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INTRODUCTION |
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The ubiquitin/26 S proteasome pathway is a major route for the selective degradation of eukaryotic proteins. Through the removal of key regulatory components, the pathway helps control many aspects of cell homeostasis, growth, and development (1-4). Examples include cell cycle progression, maintenance of chromatin structure, DNA repair, enzymatic regulation, transcription, signal transduction, and programmed cell death. In addition, the ubiquitin pathway participates in cellular housekeeping and the stress response by removing abnormal and denatured proteins.
In the ubiquitin pathway, proteins are first enzymatically tagged for breakdown by the covalent attachment of one or more chains of ubiquitin monomers. This process is catalyzed by an enzymatic cascade, involving ubiquitin-activating enzymes (E1s),1 ubiquitin-conjugating enzymes (E2s), and ubiquitin-protein ligases (E3s), that couples ATP hydrolysis to ubiquitin ligation (1-3). Attachment is via an isopeptide bond between the C-terminal glycine of ubiquitin and free lysines either in the target or in the preceding ubiquitin in the chain. Within the multiubiquitin chain, Lys48 appears to be the preferred intermolecular linkage site (5, 6) but genetic evidence has implicated several other lysines as well (e.g. Lys29 and Lys63) (7-9). Once assembled, the multiubiquitin chain functions as a recognition signal for degradation of the substrate by the 26 S proteasome, a multisubunit complex specific for multiubiquitinated proteins (10). The tagged proteins are broken down into short peptides, while the ubiquitin moieties are released intact for reuse.
Specificity within the ubiquitin pathway is achieved by at least three mechanisms. The most important determines which proteins should be ubiquitinated by the E1/E2/E3 cascade of reactions. Here, substrate specificity is primarily regulated by a large family of E2 and E3 isozymes working alone or in combination to recognize key degradation signals within the targets (1-3, 11). The second regulatory mechanism involves control of the steady-state level of ubiquitin-protein conjugates prior to breakdown. Conjugate levels are affected not only by the rate of ubiquitination but also by the rate of deubiquitination; a reaction catalyzed by a diverse family of ubiquitin-specific proteases that cleaves the junction between ubiquitin and the protein moiety (2). Through such deubiquitination, proteins can be rescued from degradation (e.g. see Refs. 12-14).
The third mechanism to achieve specificity is the association and breakdown of ubiquitin-protein conjugates by the 26 S proteasome. The 26 S proteasome is composed of two subcomplexes, designated the 19 S regulatory complex and the 20 S proteasome (4, 10, 15, 16). The 20 S proteasome contains the catalytic core of the protease; it exists as a hollow cylinder, created by the assembly of four stacked polypeptide rings, and confines the protease active sites within the lumen (17, 18). The 19 S regulatory complex binds to one or both ends of the 20 S particle. It contains ~15 subunits and confers both ATP and ubiquitin dependence to the holoenzyme complex. The function(s) of most of these subunits are unknown; six belong to the AAA family of ATPases (19, 20). Presumably, the 19 S complex recognizes appropriate targets through the multiubiquitin chain, unfolds the target moiety, and directs the unfolded polypeptide into the lumen of the 20 S complex for breakdown (4, 10). During or after this process, the multiubiquitin chain is disassembled by ubiquitin-specific proteases.
Recognition of ubiquitinated substrates by the 26 S proteasome is proposed to be mediated by one or a few common multiubiquitin chain receptors located in the 19 S particle. We recently identified a candidate for one such receptor from diverse species, designated here as Mcb1 for Multiubiquitin chain-binding protein, based on its ability to bind Lys48-linked multiubiquitin chains and its presence in the 26 S proteasome (21, 22). (Additional names include Mbp1 (21), ASF-1 and S5a (23, 24), Sun1 (25), and p54 (26) for the Arabidopsis, human, yeast (Saccharomyces cerevesiae), and Drosophila homologs, respectively.) Most of these Mcb1 polypeptides range from 376 to 414 amino acids long, the exception being yeast Mcb1 (268 amino acids), which is missing a portion of the C-terminal end present in the other Mcb1 proteins (21, 22). Consistent with a role in ubiquitin conjugate recognition, Mcb1 proteins preferentially associate with multiubiquitin chains versus the ubiquitin monomer in vitro, especially those chains containing three or more ubiquitins (21, 22, 27, 28).
Genetic studies in yeast revealed that Mcb1 may not be the only
ubiquitin receptor. This is based on the observations that yeast
Mcb1 knockout strains display a mild phenotype. Unlike
mutants in other essential ubiquitin components (2, 4),
mcb1 strains are viable, have near-wild-type growth
rates, degrade the bulk of short-lived proteins (including an
N-terminal end rule substrate) normally, and are not sensitive to UV
radiation or heat stress (22, 25). However, they are more sensitive to
amino acid analogs and cold than wild-type yeast
(22).2 In addition,
mcb1 mutants cannot degrade
ubiquitin-proline-
-galactosidase (Ub-Pro-
-gal), an artificial
substrate that is degraded by a subpathway in the ubiquitin system
(ubiquitin fusion degradation (UFD) pathway) (9). Taken together, the
results suggest that Mcb1 proteins are involved in the recognition and
degradation of only a subset of ubiquitin/26 S proteasome
substrates.
To further define the role of Mcb1 in conjugate recognition and to
identify the site(s) of multiubiquitin chain binding, we analyzed here
a series of deletion and amino acid substitution mutants of yeast and
Arabidopsis Mcb1. A stretch of highly conserved hydrophobic
residues was found to be critical for recognizing Lys48-linked multiubiquitin chains in vitro.
However, when the various Mcb1 derivatives were tested for their
ability to complement yeast mcb1, the multiubiquitin
chain recognition motif was found not to be required for conferring
resistance to amino acid analogs or for restoring degradation of
Ub-Pro-
-gal. Instead, a conserved region near the N terminus was
essential for these functions. These data show that a domain near the N
terminus, and not the multiubiquitin chain-binding site, is most
critical for Mcb1 function in vivo.
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EXPERIMENTAL PROCEDURES |
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Yeast Strains and Media--
Yeast mcb1 strain
(SV1-
MCB1; haploid MATa) was constructed previously
using strain DF5 (MATa/MAT
lys2-801/lys2-801 leu2-3, 112/leu2-3, 112 ura3-52/ura3-52 his3-
200/his3
200
trp1-1/trp1-1) (22). The congenic MATa wild-type
yeast strain (SV1) used throughout was derived from tetrad dissection
during construction of
mcb1 yeast strain. Synthetic
medium consisted of 0.7% yeast nitrogen base (Difco) supplemented with
uracil, adenine, 2% glucose, and amino acids as described previously
(29). Tryptophan was omitted from the synthetic medium for selection.
Arginine and phenylalanine were omitted from the selection medium
supplemented with canavanine and p-fluorophenylalanine, and
methionine was omitted from the synthetic medium used for
pulse-labeling experiments. Yeast transformation was carried out as
described by Gietz et al. (30), and cultures were grown at
30 °C.
Construction of the Yeast and Arabidopsis Mcb1
Mutants--
Constructions encoding the yeast and
Arabidopsis Mcb1 derivatives (see Fig. 2) were made in the
Escherichia coli expression vector pET28a (Novagen, Madison,
WI) by PCR strategies. The derived genes were verified as correct by
DNA sequence analysis. The 5-amplification oligonucleotides were
designed to add an NdeI site at the native start codon or,
for N-terminal-deleted constructions, at the deletion point to create a
new start codon. For the C-terminal-deleted constructions, the
3
-amplification oligonucleotides were designed to add an internal stop
codon. Introduced restriction sites and stop codons in the various
oligonucleotides listed below are underlined and italicized,
respectively. Positions of new start and stop codons for the
terminal-deleted constructions are indicated in Fig. 2. The PCR
products were cloned either directly or through intermediate vectors
into pET28a using the NdeI site at the 5
ends and a 3
site
which was created by the design of the 3
-amplification oligonucleotide
(EcoRI, underlined) or derived from intermediate cloning
vectors (EcoRI, HindIII, SalI, or
NdeI).
In Vitro Multiubiquitin Chain Binding Activity Assay--
All
yeast and Arabidopsis Mcb1 proteins were expressed in
E. coli strain BL21 (DE3) as His6-tagged
versions using pET28a. The His6 tag, containing the amino
acid sequence MGSSHHHHHHSSGLVPRGSH, was encoded by a nucleotide
sequence 5 to the NdeI site in the pET28a vector. Induction
of protein expression and preparation of bacterial lysates were
performed according to manufacturer's protocols (Novagen). Total
protein from cell lysates was fractionated by SDS-polyacrylamide gel
electrophoresis (PAGE) and electroblotted onto nitrocellulose membranes
(Millipore, Bedford, MA). The membranes were washed for 5 min in
Tris-buffered saline (TBS; 20 mM Tris·HCl (pH 7.5, 25 °C), 0.5 M NaCl), blocked for 2 h in TBS
containing 10 mg/ml of bovine serum albumin and washed for 5 min in
TBS. The membranes were incubated for 1-2 h at room temperature in TBS
containing 10 mg/ml bovine serum albumin and 3.5 × 105 cpm/ml of Lys48-linked multiubiquitin
chains labeled with 125I. The membranes were then washed in
TBS for 1-2 h and subjected to autoradiography. The
Lys48-linked multiubiquitin chains were prepared with
ZmUbc7 enzyme and were radiolabeled with carrier-free
Na125I (Amersham Corp.) using IODO-BEAD (Pierce) as
described previously (6).
Amino Acid Analog Sensitivity Assays--
The various
MCB1 derivatives were moved from pET28a vector into a high
copy 2µ yeast shuttle vector pRS424 (32) and expressed without the
His6 tag under the direction of the yeast ScMCB1
promoter. pRS424 was first modified to include the ScMCB1
promoter immediately followed by NdeI and EcoRI
sites. The ScMCB1 promoter, containing 552 bp upstream of
the start codon, was isolated from genomic DNA by PCR using
oligonucleotides TGCATCATTGCGAATACCGAG and
CTGTAGCTTCCAATACCATATGGCGGTTACTGC. With the exception of
the gene encoding Arabidopsis C2, the various MCB1 derivatives were moved from pET28a into the modified
pRS424 using the 5
NdeI site and a 3
EcoRI
site. Arabidopsis C
2 construction was moved using
the 5
NdeI site and a 3
SalI (filled in) into the modified pRS424 digested with NdeI and EcoRI
(filled in).
Immunological Techniques--
Yeast cultures were grown in
liquid selection medium to late logarithmic phase. Cells were collected
by centrifugation and lysed by vortexing with glass beads in 50 mM Tris-HCl (pH 8.0; 4 °C), 150 mM NaCl, 1%
Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 14 mM
-mercaptoethanol, 2 mM Na4EDTA), with the
addition of 0.5 mM phenylmethylsulfonyl fluoride and 100 nM pepstatin A just before use. Proteins were resolved by
SDS-PAGE and electroblotted onto polyvinylidene difluoride membranes
(Immobilon-P; Millipore). Immunoblot analyses were performed with
rabbit antisera against Arabidopsis or yeast Mcb1 (21, 22)
in conjunction with alkaline phosphatase-labeled goat anti-rabbit
immunoglobulins (Kirkegaard & Perry Laboratories, Gaithersburg, MD) and
the substrates nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl
phosphate.
Degradation Assays--
The plasmid directing the expression of
Ub-Pro--gal was that described by Bachmair et al. (33).
Turnover of Ub-Pro-
-gal was measured as described previously (22).
Cells were grown to exponential phase at 30 °C in synthetic medium
containing 2% raffinose, 2% galactose, and amino acids, and
pulsed-labeled for 5 min with [35S]methionine. The chase
was performed in freshly prepared synthetic medium containing 2%
raffinose, 2% galactose, amino acids, 1 mg/ml nonlabeled methionine,
and 0.5 mg/ml cycloheximide. Ub-Pro-
-gal was immunoprecipitated with
anti-
-galactosidase antibodies (Promega) and subjected to SDS-PAGE.
Radioactivity present in Ub-Pro-
-gal was quantitated by
PhosphorImager analysis.
Purification of the Yeast 26 S Proteasome--
The 26 S
proteasome was partially purified from wild-type and mcb1
yeast strains by conventional chromatography as described previously
(34). The peak of hydrolytic activity (as measured using the substrate
Suc-LLVY) from the DEAE-Affi-Gel blue column (Bio-Rad) was loaded onto
a MonoQ column (Pharmacia Biotech Inc.). Protein was eluted using a
linear gradient from 200 mM to 450 mM NaCl.
Fractions were collected and assayed for peptidase activity and the
presence of Mcb1 and Sug1/Cim3 (34). The peak of peptidase activity
eluted at around 350 mM NaCl.
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RESULTS |
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Mutational Analysis of Yeast and Arabidopsis Mcb1-- To identify domains in Mcb1 necessary for multiubiquitin chain recognition, association with the 26 S proteasome, and in vivo functions, a parallel structure/function analysis of yeast and Arabidopsis Mcb1 was initiated by constructing various deletion and amino acid substitution mutants. Amino acid sequence comparisons of Mcb1 homologs from Arabidopsis (21), Drosophila (26), humans (24), and moss3 revealed four highly conserved regions that may function in these capacities (designated domains I-IV, Fig. 1, A and B). Whereas the Drosophila, human, and moss sequences average 47% identity to Arabidopsis Mcb1 over their entire length, the four conserved domains average 72% identity. Yeast Mcb1 also contains the first three conserved domains but is lacking the C-terminal region that includes domain IV (22). Among all five proteins, domain III exhibits the greatest conservation (82% identity). It contains a short stretch of conserved hydrophobic residues (LAL/MALRL/V) surrounded by charged amino acids and is preceded by an invariant sequence GVDP (Fig. 1C). Beal et al. (36) previously proposed, from binding studies of mutant multiubiquitin chains with the 26 S proteasome, that repeated hydrophobic patches formed within assembled multiubiquitin chains are important for binding to the complex. Based on their data, we have suggested that this conserved hydrophobic patch within Mcb1 could participate in this association (21). Moreover, because Arabidopsis Mcb1 also contains a second LALAL patch near the C terminus of the protein (residues 310-314), it was possible that multiubiquitin chain binding was strengthened by the cooperative action of these motifs.
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The Conserved Hydrophobic Sequence in Mcb1 Is Critical for Binding Multiubiquitin Chains-- Evaluating the chain binding activity of the various yeast and Arabidopsis Mcb1 derivatives was facilitated by the discovery that each derivative accumulated to high levels in a soluble form when expressed in E. coli (Fig. 3A). Similar to the parental molecules (21, 22), the apparent molecular mass (as measured by SDS-PAGE) of the various deletions was substantially greater than their presumed mass. This discrepancy was especially strong for the N-terminal deletions, suggesting that the C-terminal portion has an unusual structure in the presence of SDS. The site-directed mutants that replaced all five of the LAL/MAL residues in domain III with either aspartic acid or asparagine (D5/G, N5/G, D5, and N5) (see Fig. 3) also migrated noticeably slower than the parental polypeptides.
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The N-terminal Region of Mcb1 Is Required for Amino Acid Analog
Resistance--
Although MCB1 is not essential in yeast,
deletion of this gene results in several phenotypes (22). The most
notable is an enhanced growth sensitivity to amino acid analogs,
presumably due to the accumulation of abnormal proteins that are
normally removed by the ubiquitin pathway (38). To identify region(s) of Mcb1 required for this function, we expressed a number of the yeast
and Arabidopsis Mcb1 derivatives (see Fig. 2) in the yeast mcb1 strain and examined their ability to complement the
growth defect on analog-containing medium.
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N-terminal Domain Is Required for Degradation of
Ub-Pro--Gal--
Prior phenotypic analysis of
mcb1
(22) showed that ScMcb1 is essential for the breakdown of
Ub-Pro-
-gal, a synthetic ubiquitin fusion degraded by the ubiquitin
pathway. Unlike ubiquitin fusion substrates bearing other amino acids
besides proline at the junction, the ubiquitin moiety in Ub-Pro-
-gal
is not cleaved following synthesis (33). This ubiquitin then serves as
an acceptor site for further ubiquitination by the UFD subpathway
involving the E2/E3 pair encoded by UBC4/5 and
UFD4 (9). To identify the domains within Mcb1 required for
this breakdown, the stability of Ub-Pro-
-gal was examined by
pulse-labeling in
mcb1 strains expressing various yeast
Mcb1 derivatives. Whereas Ub-Pro-
-gal was extremely stable in the
mcb1 strain, it was rapidly degraded when the
ScMCB1 gene was reintroduced
(t1/2 ~ 12 min (Fig.
8)]. This instability was similar to
that obtained with wild-type yeast and was independent of whether a
high copy 2µ (pRS424) or a low copy CEN plasmid (pRS314) was used for
expression (data not shown and Fig. 8). Each of the modifications
tested that deleted or altered the hydrophobic patch in domain III
(C
1, N5, and G) also restored rapid degradation of Ub-Pro-
-gal to
a rate nearly equivalent to that seen with ScMcb1 (Fig. 8). In
contrast, Ub-Pro-
-gal remained stable in strains expressing N
1.
These results were consistent with those obtained with the amino acid
analog sensitivity assays, suggesting that the N-terminal region
containing domain I is required for both functions.
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Assembly of Mutant Mcb1 Proteins into the 26 S Proteasome--
One
possible way to block Mcb1 function is to interfere with its
association with the 26 S proteasome. To test for such an assembly
defect, we isolated the 26 S proteasome from mcb1 strains expressing various yeast Mcb1 derivatives and assayed for the presence
of the mutant proteins. The 26 S proteasome was partially purified by
DEAE-Affi-Gel blue chromatography and then analyzed by nondenaturing
PAGE. Migration positions of the 26 S proteasome in the polyacrylamide
gels were determined by a peptidase overlay assay using the fluorogenic
20 S proteasome substrate Suc-LLVY-AMC (35). As shown in Fig.
9A, the yeast 26 S proteasome
migrates as two species (26Sa and
26Sb) on nondenaturing PAGE, corresponding to 20 S
catalytic core particles capped by two or one regulatory particles
(35).4 The mobility and
peptidase activity of both species were similar in wild-type and
mcb1 strains and in
mcb1 strains expressing yeast C
1 or N
1, indicating that none of the derivatives
substantially impaired assembly or the peptidase activity of the
holoenzyme complex.
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DISCUSSION |
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Degradation of proteins by the ubiquitin/26 S proteasome pathway
presumably requires receptors for selectively directing
multiubiquitinated proteins to the 26 S proteasome. A number of
subunits within the 19 S regulatory complex have been resolved (10),
but so far only Mcb1 has been tentatively identified as one such
receptor based on its location in the 19 S regulatory complex of the 26 S proteasome and its affinity for multiubiquitin chains (21, 22).
Although genetic analyses in yeast clearly indicates that Mcb1 cannot
be the sole ubiquitin-conjugate receptor, these studies showed that
Mcb1 has an important role in the degradation of specific ubiquitin
pathway substrates (e.g. Ub-Pro--gal) and in the
resistance to amino acid analogs (22). To identify Mcb1 domains
required for these activities, a structure/function analysis of the
yeast and Arabidopsis Mcb1 proteins was initiated. This
study was made possible, in part, by the ability of
Arabidopsis Mcb1 to complement the functions of its yeast
counterpart. Detailed mutational analysis using the
Arabidopsis and yeast homologs yielded remarkably consistent results that defined a single essential domain for multiubiquitin chain
binding in vitro. Unexpectedly, in vivo data
revealed that this domain is separate from that required for amino acid
analog resistance and Ub-Pro-
-gal degradation. Neither domain is
essential for the assembly of Mcb1 into the 26 S complex.
Analysis of both deletion and site-directed mutants localized a region
essential for multiubiquitin chain binding to a hydrophobic patch
(LAL/MALRL/V) within domain III, a highly conserved domain present in
all Mcb1 proteins (Fig. 1) (22). While it is possible that other
regions also participate, data presented here suggest that this motif
provides the major interaction site. (i) Mutants missing sequences
either C- or N-terminal to the patch (e.g. Arabidopsis C1
and C
2 and yeast N
1 and N
2) show no significant reduction in
chain binding. (ii) Deletion mutants containing just domain III flanked
by short sequences (e.g. yeast N
3 and
Arabidopsis I3 containing only 65 and 63 amino acids,
respectively) were still capable of binding multiubiquitin chains,
albeit at lower levels. (iii) Single amino acid substitutions in the
hydrophobic patch, (changing residue Leu234 (yeast) or
Val232 (Arabidopsis) to glycine) were sufficient
to dramatically impair binding. (iv) The smallest yeast and
Arabidopsis Mcb1 mutants containing only domain III had a
binding preference for multiubiquitin chains comparable to their
full-length counterparts. For Arabidopsis Mcb1 in
particular, our data eliminate a significant role for the second LALAL
patch near the C terminus of the protein (residues 310-314). A
deletion containing just this domain (N
4) failed to bind chains
whereas a deletion missing this domain (C
2) had full binding
activity by the solid-phase assay used here.
Our results are consistent with those recently published by Haracska
and Udvardy (27) who found that domain III in Drosophila Mcb1 (p54) contains a major chain-binding site. In contrast to our
work, they also detected a second chain-binding site within the
conserved domain IV, but possibly with weaker affinity. Our mutational
analysis do not support a major role for this second site. Domain IV is
not present in yeast Mcb1. Although present in Arabidopsis
Mcb1, the ability of a single amino acid substitution in domain III
(e.g. Val232 Gly) to completely block chain
binding indicates that a second site, if it is present, cannot work
autonomously. However, we cannot rule out the possibility that a second
site exists but was not detected by the different assay conditions used
here (higher temperature incubations and higher salt washes).
How the binding site in domain III interacts with multiubiquitin chains is not understood. That Mcb1 can still bind multiubiquitin chains even after SDS denaturation and fixation onto nitrocellulose membrane indicates that the binding site probably has a stable secondary structure or that it can renature upon binding to the membrane. One possibility is that the hydrophobic patch forms a pocket or loops out of the Mcb1 protein, stabilized in part by the highly charged flanking regions. This site could interact directly with complementary hydrophobic patch(es) on multiubiquitin chains, some of which may involve Leu8 and Ile44, previously shown to be required for the association of ubiquitin conjugates with the 26 S proteasome (36). Because multiubiquitin chains interact more strongly than free ubiquitin with Mcb1, we presume that the site of interaction with Mcb1 is present in the ubiquitin polymer and not found in free ubiquitin. In vitro binding studies indicate that the Mcb1 family of proteins can bind multiubiquitin chains assembled through various Lys isopeptide linkages (21, 27, 28, 37). Whether ubiquitin chains linked through lysines other than Lys48 also interact with this hydrophobic patch in Mcb1 is not known. Binding studies of the various Mcb1 mutants with alternatively linked chains and the structural solution of Mcb1/multiubiquitin complexes will be useful in this regard.
The identity of a single dominant binding site in Mcb1 is inconsistent with a model that binding involves multiple patches within Mcb1 interacting with complementary sites formed by the repeated ubiquitin monomers within the chains. It also suggests that the greater affinity of Mcb1 for longer multiubiquitin chains does not result from increased cooperative interactions among complementary repeated binding sites in both Mcb1 and multiubiquitin chains. Instead, the greater affinity could arise from the enhanced probability that one of the ubiquitin moieties will interact with domain III or from the possibility that longer multiubiquitin chains stabilize a structural motif that is absent in the ubiquitin monomer. In support of the latter, structural differences between the ubiquitin dimer, trimer, and tetramer have been noted (6, 40).
Surprisingly, analysis of the various yeast and Arabidopsis
Mcb1 mutants in yeast showed that the multiubiquitin chain-binding site
in domain III is not essential for the phenotypic functions of Mcb1,
i.e. resistance to amino acid analogs or for the ability to
degrade Ub-Pro--gal. Instead, we found that a distinct domain within
the first 60 N-terminal residues (domain I) was critical. Whereas
mutations affecting domain III could fully complement the
mcb1 deletion, mutants missing domain I failed to promote the degradation of Ub-Pro-
-gal and actually made the
mcb1 strain more sensitive to amino acid analogs.
Our ability to separate the phenotypic functions of Mcb1 from its ability to recognize multiubiquitin chains raises a question as to the role of Mcb1 in 26 S proteasome function. Because domain III mutants lack phenotypic consequences, it can be argued that the multiubiquitin chain binding activity seen in vitro is not relevant to the in vivo function of Mcb1. While definitive proof is not yet available, several observations point against this possibility. First, Mcb1 is the only subunit of purified rabbit and Drosophila 26 S proteasomes that displayed an affinity for multiubiquitin chains in vitro (27, 28). Second, the region identified here as involved in ubiquitin binding (domain III) represents the most conserved region of the molecule, implicating it as a critical domain for function. Third, both the hydrophobic nature of domain III and its preference for multimeric over monomeric and dimeric ubiquitin conforms well with predictions made concerning the specificity of a ubiquitin receptor within the 26 S proteasome (5, 28, 36). Fourth, this structural motif is highly specific for multiubiquitin binding because even single amino acid substitutions within domain III were sufficient to substantially impair binding (mutants D and G).
A more likely possibility is that Mcb1 has multiple functions. One function involves multiubiquitin chain recognition but is dispensable by virtue of the presence of other multiubiquitin receptors. The other function, which is more apparent phenotypically, is unclear but presumably requires the N-terminal region. Interestingly, a recent study has also indicated that the N-terminal domain of Mcb1 is important. It showed that human Mcb1 (or S5a) can associate both in vitro and in vivo with Id1, a helix-loop-helix protein that interacts with and blocks the action of the DNA-binding protein MyoD (41). Mcb1 appears to interact with Id1 through its N-terminal half (which includes domain I) and appears to counteract the inhibitory role of Id1 in myogenesis. How this interaction may relate to protein turnover is unclear.
What is the in vivo function of the N-terminal region? One
obvious possibility is that domain I is required for assembly of Mcb1
into the 26 S proteasome. The hypersensitivity of yeast Mcb1 mutant
N1 could be explained by its failure to integrate into the complex
while still retaining its multiubiquitin chain binding activity. The
partial loss of the hypersensitive phenotype of N
1 by affecting its
chain-binding site (N
1/G) would support this notion. However, we can
detect N
1 in the 26 S proteasome. While this association discounts a
gross defect in 26 S proteasome assembly, more subtle pertubations are
possible. Alternatively, domain I could be essential for appropriate
interactions between Mcb1 and other 19 S subunits. Potential binding
partners include Nin1 (25) and
Soi1,5 identified by genetic
screens and yeast two-hybrid analysis, respectively, as 19 S subunits
that interact with Mcb1. Expression of Mcb1 mutants missing the
N-terminal domain could interfere with proper assembly of these and
potentially other 19 S subunits into the 26 S complex. The location of
key residues in domain I as well as the identification of proteins that
interact with this region will be critical to clarify the function(s)
of Mcb1.
If Mcb1 is not the only ubiquitin receptor in the 26 S proteasome, what are other candidates? Besides Mcb1, no other 26 S proteasome subunits have been shown to have an affinity for multiubiquitin chains using the solid-phase assay (21, 27, 28). It is possible that other receptors were not identified by this assay because they failed to survive SDS denaturation and fixation onto nitrocellulose membranes or because they exist as multisubunit complexes. Both Nin1 and Soi1 have some interaction with Mcb1, but neither have been shown yet to have any ubiquitin binding activity. It is also possible that other ubiquitin receptors are not tightly associated with the 26 S proteasome and dissociate upon purification of the 19 S complex. An attractive possibility is that ubiquitin receptors freely dissociate from the 19 S complex allowing them to shuttle multiubiquitinated proteins to the 26 S proteasome. The fact that a substantial portion of cellular Mcb1 can also be found in a free form, not associated with the 26 S proteasome, is consistent with this notion (21, 22, 26). One possible candidate of this type is p62, a human phosphotyrosine-independent SH2 domain ligand, that has an affinity for ubiquitin (42). Clearly, a combination of biochemical and genetic approaches will be required to identify these additional receptor elements.
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ACKNOWLEDGEMENTS |
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We thank Drs. Quinn Deveraux and Martin Rechsteiner for help with the ubiquitin chain-binding assay, Dr. Carl Mann for supply of the Sug1/Cim3 antisera, Paul Bates for assistance in preparing radiolabeled multiubiquitin chains, Seth Davis for assistance in preparing figures, and Drs. Emily Jordan, Rich Clough, and Tanya Falbel for critical reviewing of the manuscript.
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FOOTNOTES |
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* These studies were supported in part by United States Department of Agriculture Grants NRICGP 94-37031-03347 and 97-35301-4218) and the Research Division of the University of Wisconsin-College of Agriculture and Life Sciences (Hatch 142-3936) (to R. D. V.), National Institutes of Health Grant GM43601 (to D. F.), an American Cancer Society Fellowship (to S. S.), a National Institutes of Health Fellowship (to D. M. R.), and a Damon Runyon-Walter Winchell Cancer Research Foundation Fellowship (to M. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Present address: Dept. of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706.
To whom correspondence should be addressed: Cellular and
Molecular Biology Program, Dept. of Horticulture, 1575 Linden Drive, University of Wisconsin-Madison, Madison, WI 53706. Tel.: 608-262-8215; Fax: 608-262-4743; E-mail: vierstra{at}facstaff.wisc.edu.
1
The abbreviations used are: E1,
ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3,
ubiquitin-protein ligase; Mcb1, multiubiquitin chain-binding protein,
Ub, ubiquitin; -gal,
-galactosidase; UFD, ubiquitin fusion
degradation; PCR, polymerase chain reaction; Temed,
N,N,N
,N
-tetramethylethylenediamine;
PAGE, polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; CAN, canavanine; pFP, p-fluorophenylalanine; FPLC, fast
protein liquid chromatography; AMC, 7-amino-4-methylcoumarin; bp, base pair(s).
2 H. Fu and R. D. Vierstra, unpublished results.
3 H. Fu, P. Girod, and R. D. Vierstra, unpublished data.
4 M. Glickman, D. M. Rubin, and D. Finley, manuscript in preparation.
5 K. Tanaka, personal communication.
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