From the Department of Biochemistry, University of Utah, Salt Lake City, Utah 84132
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ABSTRACT |
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The peptidase activities of eukaryotic
proteasomes are markedly activated by the 11 S REG or PA28. The three
identified REG subunits, designated ,
, and
, differ
significantly in sequence over a short span of 15-30 amino acids that
we call homolog-specific inserts. These inserts were deleted from each
REG to produce the mutant proteins REG
i, REG
i, and
REG
i. The purified recombinant proteins were then tested for
their ability to oligomerize and activate the proteasome. Both
REG
i and REG
i formed apparent heptamers and activated human
red cell proteasomes to the same extent as their full-length
counterparts. By contrast, REG
i exhibited, at low protein
concentrations, reduced proteasome activation when compared with the
wild-type REG
protein. REG
i was able to form hetero-oligomers
with a single site, monomeric REG
mutant and with REG
i. At low
concentrations, the REG
i/REG
i hetero-oligomers stimulated
the proteasome less than REG
/REG
oligomers formed from wild-type
subunits, and the reduced activation by REG
i/REG
i was due
to removal of the REG
insert, not the REG
insert. These studies
demonstrate that the REG
and REG
inserts play virtually no role
in oligomerization or in proteasome activation. By contrast, removal of
REG
insert reduces binding of this subunit and REG
/REG
oligomers to proteasomes. On the whole, however, our findings show that
REG inserts are not required for binding and activating the proteasome.
We speculate that they serve to localize REG-proteasome complexes
within cells, possibly by binding components in endoplasmic reticulum
membranes.
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INTRODUCTION |
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The proteasome is a major proteolytic organelle in the cytosol and
nucleus of eukaryotic cells (1-3). The enzyme is a cylindrical structure containing 28 subunits arranged as four rings of seven subunits each. The two end rings are composed of catalytically inactive
subunits belonging to the subunit family based on homology to
proteasome subunits from the archebacterium, Thermoplasma. The two central rings are composed of members of the
subunit family
(4-6), some of which are proteolytically active (7). Crystal
structures of Thermoplasma and yeast proteasomes reveal that
the
subunits form a central proteolytic chamber far from the
particle's surface and that entry to this central chamber is greatly
restricted (8, 9).
By itself, the proteasome does not degrade intact proteins. Association
with a 19 S regulatory complex converts the proteasome to the 26 S
protease, an energy-dependent enzyme capable of degrading intact proteins (10-13), including those marked by ubiquitin (14). The
proteasome also binds an 11 S protein complex, which we have termed REG
and others have named PA28 (15-17). REG binding to the proteasome can
stimulate peptide hydrolysis as much as 100-fold. Human red blood cell
REG is composed of two ~30 KDa subunits, REG and REG
. These two
proteins are closely related to each other and to a third protein,
REG
(18). The three proteins show extensive sequence similarities,
except for a region of 15-32 amino acids where they diverge
significantly (18, Fig. 1A). We call the divergent regions
"homolog-specific inserts" or, for sake of discussion, simply
inserts.
Using random mutagenesis, we recently isolated 45 single-site REG
mutants with impaired ability to activate the proteasome; none of the
45 amino acid changes was found in the REG
insert (19). It should be
noted, however, the REG
insert is a KEKE motif, which consists
largely of "alternating" glutamate and lysine residues and which
has been hypothesized to mediate protein-protein associations (20).
Hence, the apparent absence of single-site mutations in the REG
insert region could simply reflect redundant information within the
insert (see Fig. 1A). The crystal structure of REG
reveals that it is a heptameric ring with a conical pore measuring 20 Å in diameter on one face and 30 Å in diameter on the other (Ref. 21
and Fig. 1B). The 39 amino acids that encompass the insert
region are disordered, and the seven inserts present in the assembled
heptamer are located on the surface opposite the presumed proteasome
binding face (21). One can calculate that even if these 39 amino acids
were maximally condensed (i.e. globular), they could
interact with each other (see Fig. 1C). Furthermore, four
proteasome
subunits possess long C-terminal extensions that are
capable of interacting with REG inserts (see Fig. 1D and
Ref. 22 for a description of the C-terminal extensions). Therefore,
even though the REG
inserts appear to be some distance from the
presumed proteasome binding surface, they could interact with each
other and/or with proteasome
subunits. These interactions could, in
turn, affect REG oligomerization or the ability of REG homologs to bind
and/or activate the proteasome. To test these possibilities, we have
deleted the inserts from each REG homolog and have characterized the
three mutant proteins. At low concentrations, REG
lacking its insert
stimulates the proteasome less than wild-type REG
and produces
REG
/
hetero-oligomers that bind and activate the proteasome less
efficiently than hetero-oligomers formed from wild-type REG
and
REG
subunits. However, the biochemical properties of REG
and
REG
insert deletion mutants demonstrate that the inserts are not
required for oligomerization or activation of the proteasome by these
two REG homologs.
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EXPERIMENTAL PROCEDURES |
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Materials--
The fluorogenic peptides
t-butyloxycarbonyl
(Boc)1-Phe-Ser-Arg-aminomethylcoumarin
(FSR-MCA), Boc-Val-Leu-Lys-MCA (VLK-MCA), Suc-Leu-Leu-Val-Tyr-MCA
(LLVY-MCA), Suc-Ala-Ala-Phe-MCA (AAF-MCA), Pro-Phe-Arg-MCA (PFR-MCA),
Suc-Leu-Tyr-MCA (LY-MCA), and Cbz-Leu-Leu-Glu-NA (LLE-
NA) were
purchased from Sigma. Boc-Leu-Leu-Arg-MCA (LRR-MCA) was obtained from
Peptide International. Nitrocellulose membranes were from Schleicher
and Schuell. Peroxidase-conjugated goat anti-rabbit IgG was from
Cappel. Western blot chemiluminescence reagents were from DuPont NEN.
Site-directed mutagenesis kits were from Bio-Rad. Isopropyl-
-thiogalactopyranoside was from Boehringer Mannheim. BL-21(DE3) competent cells were from Novagen.
Construction of REG, REG
, and REG
Insert Deletion
Mutants--
Site-directed mutagenesis (23, 24) was used to produce
REG
, REG
, and REG
insert deletion mutants. The three specific DNA primers used for REG
, REG
, and REG
were
5'-GTTCACTGGGCCACAGGGAGGACCGACTGGATCAGGCACTGG-3', 5'-GGAGAAATCCACACTTAGGGACTGGAGGGTCTGGGATGGG-3', and
5'-GCATCCCATTGGGCATCACAATGGGGTCAGGGACTGG-3', respectively. To
prepare REG
, REG
, and REG
single-strand DNAs containing uracil
residues, each REG homolog cDNA was subcloned into the phagemid
pET26b and used to transform CJ236 cells, which lack dUTPase and uracil
N-glycosylase and thus allow incorporation of deoxyuridine
into DNA. A helper phage strain VCS-M13 (Stratagene) was used to infect
the CJ236 cells containing REG
, REG
, or REG
plasmids, and
single-strand DNAs were isolated according to a standard protocol (23,
24). DNAs from selected clones were purified and sequenced.
Expression and Purification of Recombinant REGi,
REG
i, and REG
i and Their Wild-type
Counterparts--
Escherichia coli BL-21 (DE3) were grown
at 30 °C overnight and induced for 2 h with 0.4 mM
isopropyl-
-thiogalactopyranoside when the
A600 was between 0.2 and 0.3 or with 0.6 mM isopropyl-
-thiogalactopyranoside when the
A600 was between 0.3 and 0.5. Lysates were
prepared by resuspending the bacteria pellets in TSD (10 mM
Tris, 25 mM KCl, 10 mM NaCl, 1.1 mM
MgCl2, 0.1 mM EDTA, 1 mM
dithiothreitol), pH 8.8, with 1 µg/ml pepstatin A and 20 µg/ml
phenylmethylsulfonyl fluoride and passing the suspension through a
French press. The lysates were then cleared by centrifugation and
applied to an ion exchange column packed with diethylaminoethyl fast
flow resin (Pharmacia Biotech Inc.). All the recombinant proteins used
in this study were purified to homogeneity using a combination of diethylaminoethyl chromatography and Superdex 200 gel filtration as
described (18, 19). Fractions from the sizing column were pooled,
concentrated with a Centriprep 10, and dialyzed against 0.5 × TSD
for at least 20 h prior to biochemical characterization.
Generation of (His)10-REGi and
(His)6-REG
i Mutants--
REG
i cDNA in
pET26b was digested with NdeI and BamHI and
ligated into pET16b to produce (His)10-REG
i
constructs. Site-directed mutagenesis was used to insert
Gly-Gly-(His)6 after the first N-terminal Met of
REG
i.
Electrophoresis--
SDS-polyacrylamide gels consisted of a 4%
stacking gel and a 15% resolving gel. Minigels prepared using a
Mini-Protean apparatus were used to separate
(His)10-REGi from REG
i and REG
(N50Y), an
inactive monomeric REG
mutant, from REG
i and to check protein purity. After gel electrophoresis, proteins were visualized using Coomassie Brilliant Blue staining or silver staining.
Hetero-oligomerization between REGi and REG
i--
To
determine whether REG
and REG
inserts affected their
hetero-oligomerization, (His)10-REG
i instead of
REG
i was used as REG
i could not be separated from
REG
i by SDS-PAGE or HPLC. Bacteria expressing
(His)10-REG
i and those expressing REG
i were
combined together and resuspended in TBS (20 mM Tris, pH 7.9, 150 mM NaCl) plus protease inhibitors (1 µg/ml
pepstatin A, 20 µg/ml phenylmethylsulfonyl fluoride). A French press
was used to lyse the bacteria, and the lysates were cleared by
centrifugation at 40,000 × g for 1 h. The lysates
were then incubated for 1 h with Ni-NTA beads equilibrated with
TBS. After discarding the supernatants, the beads were washed three
times with TBS plus protease inhibitors and three times with TBS and 25 mM imidazole plus protease inhibitors. The bound proteins
were eluted with TBS, 0.5 M imidazole and applied to a
Superdex 200 gel filtration column (see Fig. 4). To determine whether
REG
i formed hetero-oligomers with REG
i, we used
(His)6-REG
i and followed the same procedures described above; no interactions were observed between REG
i and
REG
i under these conditions.
Enzymatic Assay of the Proteasome Using Fluorogenic Substrates-- Human red blood cell 20 S proteasomes (typically 170 ng) were incubated with various amounts of wild-type or insert-deleted REG at 37 °C for 10 min in 50 µl of buffer (10 mM Tris, pH 7.5). Enzymatic reactions were started by adding 50 µl of a 200 µM solution of fluorogenic peptide substrate. After further incubation as indicated in the figure legends, the reactions were quenched with 200 µl of ice-cold ethanol, and fluorescence was measured as described (18).
Hydrolysis of Natural Synthetic Peptides-- The experiment was performed by incubating peptide P21 (SADPELALALRVSME-EQRQRQ) or BBC1 (MKKEKARVITEEEKNFKAFASLRMARANARLFGIRAKRAKEAAEQDGSG) with REG homologs or deletion mutants and human red blood cell proteasomes as described (25). The digested products were then separated on a C18 HPLC column using a gradient of 0-45% acetonitrile containing 0.1% trifluoroacetic acid.
Proteasome Binding Assay-- Proteasome binding assays were performed as described (18). Briefly, various amounts of REG homologs or hetero-oligomers were incubated with human red cell proteasomes tethered to enzyme-linked immunosorbent assay plates by the monoclonal antibody MCP20. After washing away unbound REGs, the bound proteins were eluted with 20 mM Tris, 0.5 M NaCl, pH 7.5, and dot-blotted onto a nitrocellulose membrane. The membrane was blocked with 5% nonfat dry milk in TBS, pH 7.5, 0.1% Tween 20, and bound REG proteins were detected with rabbit anti-REG followed by peroxidase-conjugated goat anti-rabbit and chemiluminescence.
Analysis of Subunit Ratio in REG/REG
Hetero-oligomers Using
HPLC--
After REG
(N50Y)/REG
and REG
(N50Y)/REG
i
hetero-oligomers were isolated from the Superdex 200 gel filtration
column as described above, three fractions across each peak were
applied to a C18 column separately, and a gradient of 30-70%
acetonitrile containing 0.1% trifluoroacetic acid was used to separate
REG
(N50Y) from REG
or REG
i. The ratio of REG
or
REG
i to REG
(N50Y) was determined by the HPLC peak area
computed by an HP ChemStation (version A. 04.01, Hewlett Packard) based
on the absorbance at 214 nm. The area of each peak was divided by the
molecular weight of the corresponding protein to produce a subunit
molar ratio.
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RESULTS |
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Purification and Oligomerization of REG, REG
, and REG
Lacking Insert Regions--
The crystal structure of REG
reveals
that the insert regions are disordered and located on the upper
structure of a heptameric REG
ring, whereas the lower surface of the
ring is the presumed proteasome binding face (Ref. 21, Fig.
1, B and C). To
address the functions of the inserts, defined by the bold
letters in Fig. 1A, we used recombinant DNA techniques
to delete them from each homolog (see "Experimental Procedures").
The mutant proteins and their wild-type counterparts were expressed in
E. coli and purified to near homogeneity (Fig.
1D). It was clear from the gel filtration step used during
purification that the REG
insert deletion mutant (REG
i) and
REG
insert deletion mutant (REG
i) formed oligomers. Nonetheless, after purification, we directly compared the
oligomerization state of each deletion mutant with its full-length
counterpart. REG
i eluted from a Superdex 200 column at 183 ml,
whereas wild-type REG
elutes at ~175 ml (data not shown).
REG
i proteins also eluted later than wild-type REG
(180 ml
versus 172 ml); REG
i eluted at 230 ml compared with
226 ml for wild-type REG
. We attribute the slight reduction in the
apparent hydrodynamic radius of REG
i and REG
i to removal of
the 28 or 32 amino acids present in the inserts rather than to changes
in the oligomerization state from a heptamer to, say, a hexamer.
However, we cannot strictly exclude the latter possibility.
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Activation of Fluorogenic Peptide Hydrolysis by REGs Lacking
Inserts--
Activation of the proteasome by each REGi was compared
with the activation properties of its full-length counterpart.
REG
i and REG
i stimulated the proteasome to the same extent
as their full-length counterparts at all protein concentrations tested (Fig. 2, A and B).
By contrast, REG
i activated the proteasome less than wild-type
REG
at low protein concentrations, although the degree of
stimulation by each REG
molecule was equivalent at high
concentrations (Fig. 2C, Table
I). Because decreased activation by
REG
i was observed using REG
and REG
i proteins obtained
in three independent purifications, we do not attribute the differences
to denaturation or variabilities in a given purification (see also Fig.
5 for similar results with REG
i/REG
i hetero-oligomers at
low protein concentrations).
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Enhanced Cleavage of Long Synthetic Peptides by REG Lacking Its
Insert--
The longest fluorogenic peptide surveyed in Table I
consists of four amino acids. Whether proteasome specificities revealed by short fluorogenic peptides are relevant to the enzyme's preference for natural peptide substrates has generated some debate (26-28). As
mentioned, the crystal structure of REG
reveals that the insert lies
on a surface opposite to the presumed proteasome binding face (21). Its
position suggests that REG inserts might bind natural peptide
substrates and either promote or retard their entry into the
proteasome's central chamber. If so, REG molecules lacking their
inserts should exhibit altered ability to promote cleavage of long,
natural peptides by the proteasome. To explore this possibility, we
used a 21-residue peptide and a 49-residue peptide as proteasome
substrates and analyzed the cleavage products in the presence of
REG
i or wild-type REG
. It is evident from the HPLC profiles in
Fig. 3 that there was no significant
difference between REG
i and REG
in their ability to stimulate
cleavage of two long peptides by the proteasome. However, we cannot
exclude the possibility that removal of the REG
insert affects the
rate at which the proteasome degrades the two substrates, P21 and BBC1. Nor can we exclude the possibility that removal of the REG
insert affects Km values for peptide hydrolysis by
REG-proteasome complexes.
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Formation of Hetero-oligomers from REG and REG
Subunits
Lacking Inserts--
REG
and REG
preferentially form
hetero-oligomers. By contrast, REG
only forms homo-oligomers (18).
The REG
and REG
inserts are similar, being composed largely of
alternating lysine and glutamic acid residues, i.e. KEKE
motifs. The REG
insert, on the other hand, contains blocks of
positive and negative amino acids sandwiched between two stretches of
hydrophobic residues (see Fig. 1A). Conceivably, the similar
chemical nature of the REG
and REG
inserts promotes preferential
association of REG
with REG
. If this were an important function
of the REG
and REG
inserts, hetero-oligomerization of
insert-deleted subunits should be impaired. This possibility was tested
by mixing (His)10-REG
i or
(His)6-REG
i with REG
i and assaying
hetero-oligomer formation by gel filtration. Bacteria that expressed
REG
i were mixed with bacteria that expressed either
(His)10-REG
i or (His)6-REG
i prior
to lysis. After clearing the lysate, the His-tagged proteins were
captured on Ni-NTA beads, eluted with 0.5 M imidazole, and applied to a Superdex 200 filtration column (Fig.
4, A and B). (His)10-REG
i formed active, stable hetero-oligomers
with REG
i, whereas (His)6-REG
i did not (data
not shown). To rule out the possibility that association of
(His)10-REG
i with REG
i was due to the
interactions between REG
i and the polyhistidine extension on
REG
i, we used a REG
inactive monomeric mutant, REG
(N50Y), that forms hetero-oligomers with wild-type
REG
.2 REG
(N50Y) was
mixed with REG
i, and gel filtration revealed that the two
proteins formed stable active hetero-oligomers (Fig. 4, C
and D). From these experiments, we conclude that the
homolog-specific inserts do not determine the preferential association
of REG
and REG
.
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Activity of REG/
Hetero-oligomers Formed from Subunits
Lacking Inserts--
The formation of stable hetero-oligomers between
REG
i and REG
i allowed us to measure the ability of this
complex to activate the proteasome and compare it with activation by
wild-type REG
/REG
hetero-oligomers. As shown in Fig.
5A, higher concentrations of REG
i/REG
i were needed to activate the proteasome to the
same extent as wild-type REG
/REG
oligomers. Reduced proteasome
activation at low concentrations of REG
i/REG
i might result
from the lack of the REG
insert, the REG
insert, or both. To
distinguish among these possibilities, we measured proteasome
activation by hetero-oligomers of REG
(N50Y)/REG
or
REG
(N50Y)/REG
i and found that REG
(N50Y)/REG
i produced
less activation than REG
(N50Y)/REG
at low concentrations (Fig.
5B). By contrast, REG
i/REG
activated the proteasome
in a manner indistinguishable from REG
/REG
(Fig. 5C).
Therefore, the reduced stimulation by REG
i/REG
i can be
attributed to the absence of the REG
insert, not the REG
insert.
This result is consistent with the impaired ability of REG
i to
stimulate the proteasome (Fig. 2C). Apparently, the REG
insert affects proteasome activation even in the REG
/REG
hetero-oligomer.
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DISCUSSION |
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The experiments presented above address four hypotheses regarding
the functions of REG homolog-specific inserts. We previously hypothesized that KEKE motifs promote protein-protein association (20).
More recently, we proposed that the KEKE motifs in REG and REG
might interact with KEKE motifs on the proteasome subunits C6 and C9
(18). The geometric considerations presented in Fig. 1D show
that even though the KEKE inserts in REG
are located on the surface
opposite to the presumed proteasome binding surface, they could still
interact with C-terminal extensions on proteasome alpha subunits. Thus,
it is reasonable to imagine that REG inserts might promote binding to
the proteasome, and this constitutes the first hypothesis tested. Fig.
2, A and B, demonstrates that proteasome
activation by REG
and REG
deletion mutants are virtually identical to their wild-type counterparts, indicating that binding of
REG
or REG
to the proteasome is not impaired by removal of their
corresponding inserts. Although REG
and REG
inserts do not
promote proteasome binding, several experiments show that the REG
insert is important for binding to the proteasome. First, the results
in Fig. 2C demonstrate that, at low concentrations, REG
i stimulates the proteasome much less than wild-type REG
. Likewise, low concentrations of REG
i/REG
i hetero-oligomers activate the proteasome to a lesser extent than REG
/REG
hetero-oligomers (Fig. 5A). Impaired proteasome activation
by REG
i/REG
i was due to removal of REG
inserts because
REG
i/REG
hetero-oligomers stimulate the proteasome equally as
well as REG
/REG
do at all concentrations tested (Fig.
5C). REG
(N50Y)/REG
i, on the other hand, stimulates
the proteasome less than REG
(N50Y)/REG
at low concentrations
(Fig. 5B). Reduced proteasome activation by REG
i and
REG
i/REG
i could reflect weaker binding to the proteasome; indeed, a direct proteasome binding assay revealed that
REG
(N50Y)/REG
i binds less tightly than REG
(N50Y)/REG
(Fig. 5D). It is possible that the reduced activity of
REG
i results from structural alterations in the subunit produced
by removal of the insert. However, in view of the high degree of
sequence homology surrounding the
,
, and
inserts (Fig.
1A), it seems unlikely that structural perturbations
produced by deleting the inserts would be confined to the
subunit.
For this reason, we favor the idea that the REG
insert actively
contributes to proteasome binding by this subunit and by
hetero-oligomers containing this subunit. Still, we must admit that the
reduced activation by REG
i and hetero-oligomers formed from
REG
i can also be explained by subtle structural alterations of
the REG
subunit upon removal of its insert.
The second hypothesis asked whether the inserts are responsible for the
fact that REG and REG
activate cleavage of a variety of
fluorogenic substrates, whereas REG
mostly activates cleavage after
basic amino acids (18). The results presented in Table I show that the
patterns of peptide cleavage are identical regardless of whether
full-length REG homologs or their insert-deleted counterparts were used
to activate the proteasome. It is clear, therefore, that the
homolog-specific inserts do not determine the patterns of activated
fluorogenic peptide cleavage by the proteasome. The unique pattern of
activation by REG
, as opposed to REG
and REG
, may be defined
by other region(s) in the molecules, e.g. C-terminal and/or
N-terminal sequences. Future experiments in which these regions are
exchanged between REG
and REG
should provide answers to this
question.
The crystal structure of REG reveals that the inserts surround a
pore through the REG
heptamer (Fig. 1, B and
C). Because of their location, the inserts could bind long
peptides and promote entry of these substrates to the active sites of
the proteasome. Alternatively, the inserts might prevent longer
substrates from being degraded by blocking the pore of REG
or by
directly binding longer peptides. These two possibilities constitute
the third hypothesis tested. The HPLC profiles in Fig. 3 show that
there are no significant differences in the ability of REG
and
REG
i to stimulate the digestion of two long peptide substrates by
the proteasome. However, we consistently observe that REG
i
stimulates the proteasome slightly better than wild-type REG
using
both Suc-LLVY-MCA and long peptides as substrates (Fig. 2A,
Fig. 3, and data not shown). Because the differences are within the
experimental error, they may simply result from experimental variation.
In any event, removal of the REG
insert produces, at most, a small effect on substrate degradation.
The last hypothesis concerns the possibility that the inserts are
responsible for the fact that REG preferentially binds REG
, and
neither of these subunits binds REG
(18). The results presented in
Fig. 4 clearly rule out a requirement for REG
and REG
inserts in
hetero-oligomer formation. While this manuscript was in review, a study
from DeMartino's laboratory (29) reported that deletion of the insert
from rat REG
does not affect its activity, a finding that agrees
with results presented here. However, Song et al. (29) found
reduced activation by REG
/REG
hetero-oligomers formed from rat
REG
molecules lacking inserts, whereas removal of the human REG
insert did not affect proteasome activation by REG
/REG
(Fig.
5C). Song et al. (29) speculate that decreased activation resulted from impaired interactions between REG
and REG
upon removal of the REG
inserts. Although we cannot rule out
the possibility that the inserts facilitate association of these two
subunits, they are clearly not required for human REG
to oligomerize
with REG
(see Fig. 4). Song et al. (29) also reported
that recombinant rat REG
is inactive. The results presented here
(Fig. 2C and Table I) show that recombinant human REG
stimulates the proteasome. Two other studies from our laboratory have
shown that human REG
is active (18, 19). Moreover, a single-site mutation in the activation region of REG
results in loss of this activity (19). Therefore, we do not believe that the activity observed
for human REG
is an experimental artifact. The discrepancy between
our findings and those of Song et al. (29) could reflect differing properties of rat and human REG
molecules or differences in assay conditions. Regarding the latter possibility, we note that the
assay of Song et al. uses REG
concentrations 10-fold lower than those employed by us.
In summary, the results presented above clearly demonstrate that the
presence of REG inserts increases the affinity of REG
and
REG
/REG
hetero-oligomers for the proteasome. By contrast, REG
and REG
inserts are not required for proteasome binding or
activation. We have also demonstrated that the REG
, REG
, and
REG
inserts are not responsible for their differing abilities to
activate cleavage of specific fluorogenic peptides, nor do the inserts
define the specific patterns of homo-oligomerization and/or the
hetero-oligomerization observed with these three homologs. In addition,
removal of the REG
inserts did not have any pronounced effect on the
extent of the degradation of long peptides. If the REG
and REG
inserts are not important for activating the proteasome, what do they
do? Although it is possible that they have no biological functions, the
high degree of conservation observed among each homolog-specific insert
from several mammalian species would suggest otherwise. Possibly, the
inserts have some regulatory function. For example, REG
has been
reported to disappear upon interferon-
treatment (30), raising the
possibility that the REG
insert harbors a degradation signal. As for
REG
and REG
, we have observed that KEKE motifs are found in
chaperonins such as hsp90 and hsp70 (20). Conceivably, the KEKE insert
of REG
may bind heat shock proteins. Alternatively, we have proposed
that the inserts serve to couple the proteasome to the calnexin-TAP-MHC
I complexes in the endoplasmic reticulum, thereby promoting transfer of
antigenic peptides to MHC I molecules (20). The availability of
insert-deleted forms of each REG homolog should allow direct tests of
the latter hypothesis.
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ACKNOWLEDGEMENTS |
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We thank Robert Schackmann for oligonucleotide synthesis. We are also grateful to Drs. Carlos Gorbea, Patrick Young, Vicenca Ustrell, David Mahaffey, Laura Hoffman, and Daniel Taillandier for comments on the manuscript and to other members of the Rechsteiner laboratory for technical advice.
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FOOTNOTES |
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* These studies were supported by National Institutes of Health Grant GM37009 and by grants from the Lucille P. Markey Charitable Trust and the American Cancer Society.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.
1
The abbreviations used are: Boc,
t-butyloxycarbonyl; NA,
-naphthylamide; Cbz,
benzyloxycarbonyl; HPLC, high performance liquid chromatography; MCA,
aminomethylcoumarin; MHC, major histocompatibility complex; REG, 11 S
regulator; Suc, succinyl; TAP, transporter associated with antigen
processing; PAGE, polyacrylamide gel electrophoresis.
2 Z. Zhang, A. Clawson, and M. Rechsteiner, manuscript in preparation.
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REFERENCES |
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