(Received for publication, August 23, 1995; and in revised form, October 27, 1995 )
From the
The activity of the intracellular protease, the proteasome, is
modulated by a number of specific regulatory proteins. One such
regulator, PA700, is a 700,000-Da multisubunit protein that activates
hydrolytic activities of the proteasome via a mechanism that involves
the ATP-dependent formation of a proteasome-PA700 complex. Four
subunits of PA700 have been shown previously to be members of a protein
family that contains a consensus sequence for ATP binding, and purified
PA700 expresses ATPase activity. We report here the identification,
purification, and initial characterization of a new modulator of the
proteasome. The modulator has no direct effect on the activity of the
proteasome, but enhances PA700 activation of the proteasome by up to
8-fold. This activation is associated with the formation of a
proteasome/PA700-containing complex that is significantly larger than
that formed in its absence. The modulator has a native M of
300,000, as determined by gel filtration chromatography,
and is composed of three electrophoretically distinct subunits with M
values of 50,000, 42,000, and 27,000 (p50, p42,
and p27, respectively). Amino acid sequence analysis of the subunits
shows that p50 and p42 are members of the same ATP-binding protein
family found in PA700. The p50 subunit is identical to TBP1, a protein
previously reported to interact with human immunodeficiency virus Tat
protein (Nelbock, P., Dillion, P. J., Perkins, A., and Rosen, C.
A.(1990) Science 248, 1650-1653), while the p42 subunit
seems to be a new member of the family. The p27 subunit has no
significant sequence similarity to any previously described protein.
Both p50 and p42, but not p27, were also identified as components of
PA700, increasing the number of ATP-binding protein family members in
this complex to six. Thus, p50 and p42 are subunits common to two
protein complexes that regulate the proteasome. The PA700-dependent
proteasome activator represents a new member of a growing list of
proteins that regulate proteasome activity.
The proteasome is a 700,000-Da multicatalytic protease that
participates in a number of proteolytically mediated intracellular
processes, including the constitutive turnover of many intracellular
proteins(1) , the rapid elimination of proteins with abnormal
structures(2, 3) , the temporal reduction in levels of
critical regulatory proteins for control of the cell cycle and
transcription (4, 5, 6, 7) , the
proteolytic activation of the transcription factor NF-B (8) , and the processing of antigens for presentation by class
I major histocompatibility complex proteins(9, 10) .
Despite the important role of the proteasome in these various
processes, the mechanisms by which its action is controlled remain
unclear. Several lines of evidence indicate that proteasome function is
controlled by specific regulatory proteins. First, the proteasome can
be isolated as part of a larger protein complex (M
1,500,000) referred to as the ``26 S
protease''(11) . This complex displays catalytic and
regulatory properties that differ considerably from those of the
purified 20 S proteasome, most likely because of regulatory influences
exerted by the non-proteasome components of the complex. Second,
individual proteasome regulatory proteins have been identified and
purified. One of these proteins, which we call PA700 and which has been
independently described in several laboratories, appears to represent
the major non-proteasome component of the 26 S
protease(10, 12, 13, 14) . PA700 is
a 700,000-Da multisubunit ATP-dependent proteasome activator. It forms
a complex with the proteasome by a mechanism that requires ATP
hydrolysis. The proteasome-PA700 complex has physical properties, such
as molecular weight, and catalytic properties, such ATP-dependent
degradation of ubiquitinated proteins, that are characteristic of the
purified 26 S protease. At least four of the
20
electrophoretically distinct subunits of PA700 are homologous to one
another and are members of a large protein family that contains a
consensus sequence for ATP
binding(15, 16, 17) . Some of these same
proteins have been identified as components of the purified 26 S
protease, providing additional strong evidence that the
proteasome-PA700 complex is similar, if not identical, to the 26 S
protease(13, 15, 16) . One or more of these
ATP-binding proteins may be responsible for the function of ATP in
proteasome activation. In fact, purified PA700 expresses ATPase
activity. Surprisingly, many of these ``ATPase'' subunits of
PA700 have been identified independently as proteins involved in
processes with no obvious relationships to proteasome
function(17) . These findings may be explained by new and
unexpected roles for the proteasome or may indicate that a given ATPase
protein has multiple cellular functions.
During the course of our continuing characterization of the function of PA700, we have identified a new protein complex that functions as a PA700-dependent activator of the proteasome. This report describes the identification, purification, and initial structural and functional characterization of this protein, which contains two members of the ATPase protein family. Furthermore, the same two proteins are identified here as new subunits of PA700, raising the number of family members in this complex to six.
Figure 1:
Identification of a PA700-dependent
activator (modulator) of the proteasome by gel filtration
chromatography. Proteins from Fraction II that precipitated between 0
and 38% saturated ammonium sulfate (see ``Materials and
Methods'') were solubilized and chromatographed on Sephacryl
S-300. Column fractions were assayed for PA700 activity () and
PA700-dependent activator (modulator) activity (
) as described
under ``Materials and Methods.'' PA700 activity was assessed
using 5 µl of column fractions and the purified exogenous 20 S
proteasome (0.25 µg/assay, 0.4 units). Modulator activity was
assessed using 5 µl of column fractions and the purified exogenous
proteasome (0.25 µg/assay) and purified exogenous PA700 (0.64
µg/assay, 7.6 units). Control assays for the endogenous proteasome
in column fractions had <1.5 units/assay (not shown). The column
fractions were also subjected to Western blotting with anti-TBP1 (top panel). To show all fractions, results from two different
blots are shown. Standards of TBP1 on each blot produced bands of equal
intensity.
Figure 2: Ion-exchange chromatography of the PA700-dependent proteasome activator (modulator). Column fractions from the Sephacryl-S300 column (see Fig. 1) containing modulator activity (fractions 110-125) were pooled and subjected to ion-exchange chromatography on DEAE-Fractogel as described under ``Materials and Methods.'' Column fractions were assayed for PA700-dependent activator (modulator) activity. Samples (5 µl) of the column fractions were assayed in the presence of the purified exogenous proteasome (0.25 µg) and PA700 (0.67 µg, 8.8 units).
The fractions containing the
PA700-dependent proteasome-activating activity were pooled and
subjected to further purification by ion-exchange chromatography on
DEAE-Fractogel as described under ``Materials and Methods.''
The PA700-dependent proteasome-activating activity bound to this resin
and was eluted as a single peak at a position corresponding to 100
mM NaCl (Fig. 2). The fractions containing the peak
activities were pooled and subjected to hydroxylapatite column
chromatography. The PA700-dependent proteasome-activating activity
eluted from this resin as a single peak (Fig. 3). SDS-PAGE
analysis of the fractions from the hydroxylapatite column showed that
three major proteins (denoted with arrows in Fig. 3)
had elution profiles indistinguishable from one another and were
coincident with PA700-dependent proteasome activation. These proteins
had apparent M
values of 50,000, 42,000, and
27,000. Retrospective analysis of the column fractions from the
Fractogel ion-exchange chromatography by SDS-PAGE also showed that
these three proteins coeluted with one another and with modulator
activity (data not shown). The activity from the hydroxylapatite
chromatography was subjected to a second Sephacryl S-300 chromatography
step. The activity eluted at a position corresponding to its originally
estimated M
of 300,000 and was coincident with the
three proteins described above (data not shown). Therefore, we conclude
that red blood cell extracts contain a PA700-dependent proteasome
activator composed of protein subunits with M
values of 50,000, 42,000, and 27,000.
Figure 3: Hydroxylapatite chromatography of the PA700-dependent proteasome activator (modulator). Column fractions from the DEAE-Fractogel column (see Fig. 2) containing modulator activity (fractions 14-21) were pooled and subjected to hydroxylapatite chromatography as described under ``Materials and Methods.'' Column fractions were assayed for PA700-dependent activator (modulator) activity. Samples (5 µl) of the column fractions were assayed in the presence of the purified exogenous proteasome (0.25 µg) and PA700 (0.67 µg, 5.3 units). Inset, column fractions were subjected to SDS-PAGE. Three protein bands (p50, p42, and p27), denoted with arrows, had elution profiles similar to one another and modulator activity. In this figure, the p27 protein did not reproduce well, although it was clearly visible on the original gel.
Figure 4:
Effect of the modulator on proteasome
activity. The purified modulator was tested for its ability to activate
the purified proteasome. The indicated amounts of modulator were
preincubated for 45 min with 0.25 µg of proteasome in the presence
() or absence (
) of PA700 (0.64, 2.5, or 5.0 µg/assay)
and in the presence of 100 µM ATP prior to the assay for
proteasome activity using succinyl-Leu-Leu-Val-Tyr
7-amino-4-methylcoumarin as a substrate.
Figure 5:
Effect of the modulator on
proteasome-containing complexes isolated by glycerol density gradient
centrifugation. Various combinations of proteins were subjected to
glycerol density gradient centrifugation as described under
``Materials and Methods.'' The purified modulator (14.6
µg) was centrifuged, and 15 µl of the fractions were assayed in
the presence of 0.25 µg of proteasome and 0.67 µg of PA700
(). The purified proteasome (10.2 µg) was centrifuged, and 40
µl of each fraction were assayed (
). The peak fraction,
number 12, had 0.5 units of activity. The proteasome (10.2 µg) and
PA700 (35 µg) were preincubated and centrifuged. 40 µl of the
fractions were assayed for proteasome activity (
). The
proteasome (10.2 µg), PA700 (35 µg), and the modulator (35
µg) were preincubated and centrifuged. 40 µl of the fractions
were assayed for proteasome activity
(
).
Figure 6: The p50 subunits of the modulator and PA700 are identical to one another and to TBP1. The p50 subunit of the modulator was isolated by HPLC and SDS-PAGE and subjected to amino acid sequencing as described under ``Materials and Methods.'' The sequences of 12 peptides produced by Lys-C digestion were determined and are shown aligned with the complete sequence of human TBP1(21) . The p50 subunit of PA700, identified by its reactivity to an anti-TBP1 antibody, was isolated and subjected to sequencing in the same manner as the modulator subunit. The sequences of five peptides were determined and are shown aligned with TBP1.
Figure 7: The p42 subunits of the modulator and PA700 are identical to one another and are homologous to p50 (TBP1). The p42 subunit of the modulator was isolated by HPLC and SDS-PAGE and subjected to amino acid sequencing as described under ``Materials and Methods.'' The sequences of five peptides produced by Lys-C digestion were obtained and are shown aligned with peptides from p50. A dash denotes an identical amino acid at a given position. The p42 subunit of PA700 (isolated as described under ``Materials and Methods'') was subjected to amino acid sequencing, and three peptides (denoted by asterisks) had identical sequences to the corresponding peptides from p42 of the modulator.
Figure 8: The anti-TBP1 antibody cross-reacts with a subunit of both the modulator and PA700. Left panel, PA700 (2 µg) and the modulator (0.6 µg) were subjected to SDS-PAGE and stained with Coomassie Blue; right panel, PA700 (4 µg) and the modulator (1 µg) were subjected to immunoblotting with an antibody against TBP1 as described under ``Materials and Methods.'' Protein standards are indicated.
Figure 9:
Identities of PA700 subunits, including
six members of the ATP-binding protein family. Purified PA700 was
subjected to two-dimensional analysis as described under
``Materials and Methods.'' The individual peaks resolved by
HPLC (peaks 1-12) were electrophoresed on a
SDS-polyacrylamide gel. Individual subunits whose identities are
established, including the six members of the ATP-binding protein
family, are denoted with arrows and are as follows. peak1: upper band, S4(22) , and lower
band, p45(15, 26) ; peak 2:
MSS1(23, 24) ; peak 3: p40 (Mov34) (7, 33, 34) ; peak 4: p50
(TBP1)(21, 35) ; peak 5: upper band,
TBP7(15, 25, 35) , and lower band,
p42 (this report); peak 6: p58
(P91A)(15, 36) ; peak 10: p31
(Nin1p)(6) ; peak 11: upper band, p112
(Sen3p) (15) (GenBank accession number L06321),
and lower band, p97(15, 16) ; peak
12: p44 (HUMORF07) (16) .
Figure 10: Comparison of modulator and PA700 subunits by SDS-PAGE. Subunits from the modulator and PA700 were compared by SDS-PAGE. Lanes 1 and 5, 1.5 µg of purified modulator; lanes 2-4, PA700 subunits isolated by HPLC: lane 2, p50 subunit (peak 4 from Fig. 9); lane 3, p42 subunit (peak 5 from Fig. 9); lane 4, peak 9 from Fig. 9showing no common band with p27 from the modulator.
We have identified a new multisubunit protein complex that regulates proteasome function. Unlike previously identified proteasome regulators, this new protein, referred to here as modulator, does not directly influence proteasome activities, but enhances, by up to 8-fold, the effect of PA700, an ATP-dependent proteasome activator. Because the modulator's stimulatory effect magnifies a 50-200-fold stimulatory effect by PA700, its influence on total proteasome activity is very large. The mechanism by which the modulator exerts its effect is presently unclear. PA700 activation of the proteasome involves the formation of a proteasome-PA700 complex in which PA700 binds to one or both of the proteasome's terminal rings. The finding that the modulator promotes the formation of a larger complex than that which is formed in its absence (Fig. 5) suggests two possible mechanisms for its action, which are not mutually exclusive and do not represent all possible mechanisms. First, the modulator might form a ternary complex with PA700 and the proteasome. Alternatively, the modulator could promote the formation of more complexes in which the proteasome is bound to two, rather than just one, PA700 molecule. Each of these models would account for the larger proteasome-containing complex caused by the modulator, and structural changes associated with each presumably result in increased proteasome activation.
Two modulator subunits, p50 and p42, are homologous to one another and are members of a large protein family that contains a consensus sequence for ATP binding(17) . The p50 subunit is identical to TBP1, previously identified as a human immunodeficiency virus Tat-binding protein(21) , while p42 seems to be a new family member. We are currently determining the complete primary structure of p42 to confirm this latter conclusion. In any case, this work demonstrates that p50 and p42 modulator subunits are also subunits of PA700. Four other members of this protein family, S4, MSS1, p45, and TBP7, previously were shown to be subunits of PA700. Thus, the results presented here demonstrate that PA700 contains at least six members of this ATPase family.
The surprising finding that p50 and p42 are
common subunits of two distinct proteasome regulators raises obvious
questions regarding the origin of the modulator protein, on the one
hand, and the basis for the identification of p50 and p42 as PA700
subunits, on the other. For example, could the modulator represent a
subcomplex of PA700, derived from the dissociation of PA700? To address
this question, we attempted to generate the modulator from purified
PA700 by treating purified PA700 (which contains p50 and p42 as major
components as judged by staining intensity; Fig. 9) with a
variety of chaotropic and other agents (including 38% ammonium sulfate)
and then subjecting the protein to gel filtration chromatography or to
density gradient centrifugation. The p50 and p42 subunits of PA700
always migrated coincidently with PA700 activity and with the rest of
the PA700 subunits, and we never observed formation of the modulator
from PA700 in these experiments. ()The possible origin of
the modulator as a dissociated subcomplex of PA700 also would imply the
existence of a complementary PA700 subcomplex devoid of p50 and p42 and
suggests that modulator activity might represent a
``reconstitution'' effect, i.e. that the restoration
of critical subunits to a functionally incompetent form of PA700
restores PA700 activity. We attempted to detect such PA700 species in
our preparations by fractionating purified PA700 by gel filtration or
glycerol density gradient centrifugation and then assaying the
fractions for PA700 activity and for the ability to be stimulated by
the purified exogenous modulator. In all cases, both of these
activities were exactly coincident with one another and with all PA700
subunits (including p50 and p42) detected by SDS-PAGE. Such results
would not be expected if the modulator had a selective effect on a
subpopulation of PA700 that lacked p50 and p42 because such a
population should have a lower molecular weight than native PA700.
Thus, we have been unable to provide any evidence that the modulator is
derived from dissociation of PA700, although our current analysis
cannot rigorously exclude this possibility.
Could the identification of p50 and p42 in PA700 preparations represent contamination by the modulator, either nonspecifically or as the result of a specific interaction between PA700 and the modulator? Nonspecific contamination seems unlikely because PA700 and the modulator were well separated from each other early in the purification (Fig. 1) and differed significantly in their chromatographic behavior on several additional columns. Furthermore, both p50 and p42 represented major PA700 components (Fig. 9). Although we cannot completely exclude the possibility that our PA700 preparations contain complexes formed by the specific interaction of the modulator and PA700, the failure to dissociate the modulator from PA700 preparations (as described above) seems to argue against this possibility. Therefore, these various results support the conclusion that the modulator represents a distinct protein complex that shares two subunits, p50 (TBP1) and p42, with PA700.
The sharing of p50 and p42 between two different proteins suggests that these subunits could have functions common to each of the complexes. Because p50 and p42 are members of the ATP-binding protein family, such a function might involve the role of ATP in proteasome activation, particularly in the assembly of the proteasome-containing complex. We are currently examining this possibility. In any case, another member of the ATP-binding protein family, p45, has been identified in at least two different multiprotein complexes: PA700 (15) and the transcriptional mediator complex that contains Sug1p, the homolog of p45 in yeast(26, 27, 28) . It seems possible that other members of this ATP-binding protein family will be shared among different protein complexes.
The regulation of proteasome function
by the combined action of two proteins, as shown here, is reminiscent
of early reports by Hershko and co-workers (29) showing the
requirement for two factors, termed CF1 and CF2, for proteasome
activation. Therefore, it is reasonable to question the possible
relationship between PA700 and the modulator, on the one hand, and CF1
and CF2, on the other. Direct comparison between these protein pairs is
difficult because CF1 and CF2 were not purified. Nevertheless, as
discussed previously, we believe that CF1 is structurally and
functionally related, but not identical, to PA700 (12) . In
contrast, there are significant differences between the modulator and
CF2. Goldberg and co-workers (30) purified a protein with
functional properties indistinguishable from CF2 (i.e. the
ability to activate the proteasome in a CF1-dependent manner in a
reconstitution system where proteasome activation required both
proteins). In the absence of CF1, this CF2 protein functioned as a
proteasome inhibitor and was judged identical to a previously
identified proteasome inhibitor with a subunit size of 40,000
Da(31) . We have failed to detect inhibition of the proteasome
by the modulator under a variety of assay conditions. Furthermore, the 40,000-Da inhibitor was subsequently shown to
have the same structural, functional, and immunological properties as
-aminolevulinic-acid dehydratase(32) . These various
findings clearly distinguish the modulator described here from
previously described CF2 proteins and suggest that the modulator may be
one of several proteins that regulate proteasome function indirectly
through PA700.