From the William S. Rowe Division of Rheumatology,
Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio 45229 and the § Department of Molecular Genetics, University of
Cincinnati College of Medicine, Cincinnati, Ohio 45267
Received for publication, September 11, 2002, and in revised form, November 26, 2002
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ABSTRACT |
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Vertebrate proteasomes are structurally
heterogeneous, consisting of both "constitutive" (or
"standard") proteasomes and "immunoproteasomes." Constitutive
proteasomes contain three ubiquitously expressed catalytic subunits,
Delta ( Eukaryotic proteasomes are an integral component of
ubiquitin-mediated protein degradation, which plays a major role in the turnover of cytoplasmic and nuclear proteins (1-5). By virtue of their
role in protein metabolism, proteasomes are involved in a number of
cellular processes, including cell cycle control, cellular stress
responses, intracellular signaling, and major histocompatibility
complex class I antigen processing (6). Immunoproteasomes are a
specialized subset of vertebrate proteasomes that contain three
interferon- The 20 S catalytic proteasome core is comprised of 28 subunits arranged
in four stacked seven-member rings (15-17). Each outer ring contains
seven different non-catalytic Proteasome assembly is a slow process that involves detectable
intermediate complexes with half-lives of several hours (such as 5-6 h
in H6 cells) (27). These "preproteasome" intermediates contain one
complete An important question is what happens to proteasome diversity when both
types of catalytic subunits are expressed in the same cell, which is
the case in cells of the immune system and other cells under the
influence of interferon- In the current study, we demonstrate that differences between the MECL
( DNA Constructs--
The cDNAs encoding human MECL and Z were
expressed in T2 cells using an episomal vector, pCEP4, which confers
hygromycin resistance (Invitrogen). Propeptide switches were carried
out using KpnI restriction sites that were engineered at the
site of propeptide cleavage between Gly Antibodies--
Polyclonal antisera recognizing LMP2, LMP7,
MECL, Delta, Z, and C8 were obtained from rabbits immunized with
recombinant mouse subunits. Anti-proteassemblin is a rabbit polyclonal
antiserum raised against recombinant human proteassemblin. Anti-X
(P93250) is a rabbit polyclonal antiserum raised against human X that
was obtained from K. B. Hendil (August Krogh Institute, University of Copenhagen, Copenhagen, Denmark). Anti-N3, obtained from K. B. Hendil, is a mouse monoclonal antibody that recognizes human N3
(34). Anti-Myc and anti-FLAG are mouse monoclonal antibodies that respectively recognize a Myc epitope, EQKLISEEDL (Invitrogen), or
a FLAG epitope, DYKDDDDK (Sigma).
Cell Culture and Transfection--
Lymphoblastoid T2 cells (35)
obtained from P. Cresswell (Yale University School of Medicine, New
Haven, CT) were grown in RPMI 1640 medium supplemented with 10% fetal
bovine serum, L-glutamine, and antibiotics (R10) as
described (31). Episomal expression vectors (pCEP4) containing MECL or
Z constructs were transfected by electroporation (250 V and 500 microfarads) in serum-free RPMI at 2.5 µg of DNA/5 × 106 cells. Transfected cells were grown in R10 for 48 h before the addition of hygromycin at 360 units/ml (Calbiochem).
T2-derived stable transfectant cell lines were generated by
co-transfecting pSV2.Neo (2.5 µg, Clontech) with
pSG5 vectors (25 µg, Stratagene) containing proteasome subunit
cDNAs. Stable transfectant clones were selected with 0.8 mg/ml of active G418 (Invitrogen). Selected cells were
maintained in RPMI with 5% bovine serum (R5) with either G418 for
pSV2.Neo and/or hygromycin for pCEP4.
Anti-FLAG Immunoprecipitation, Gel Electrophoresis, and
Immunoblotting--
Cells were lysed with 1% Nonidet P-40 in 20 mM Tris, pH 7.6, 10 mM EDTA, and 100 mM NaCl. FLAG-tagged proteasome subunits and their
associated complexes were immunoprecipitated from post-nuclear supernatants with anti-FLAG-M2-Sepharose (Sigma), subjected to 12.5% SDS-PAGE, and transferred by electroblotting onto polyvinylidene difluoride paper at 500 mA for 60 min. Co-precipitating proteins were detected using specific antisera (anti-LMP2, anti-LMP7, anti-Z, anti-Delta, anti-X, anti-N3, and anti-proteassemblin) or monoclonal antibodies (anti-FLAG and anti-Myc) as primary antibodies, alkaline phosphatase-conjugated goat anti-rabbit or goat anti-mouse secondary antibodies, and 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (Sigma) for color development.
MECL Metabolic Labeling, Anti-C8 Immunoprecipitation, and
Two-dimensional Gel Electrophoresis--
Splenic ConA blasts (10 × 106 cells) were pulse-labeled with 0.25 mCi of
[35S]methionine/cysteine for 45 min. One-half of the
cells then were lysed with 0.5% Nonidet P-40; the rest were chased for
4 h in R10 containing 4 mM cold methionine and then
were lysed. Labeled preproteasomes were immunoprecipitated with
anti-C8 from post-nuclear lysates, eluted in a non-equilibrium pH
gradient electrophoresis (NEPHGE) sample buffer, and subjected to
two-dimensional NEPHGE-PAGE and autoradiography (27).
The Z Propeptide Enables MECL Incorporation into Proteasomes That
Contain Delta and X and Facilitates Incorporation of X into
MECL+/LMP2+ Proteasomes--
The
inducible proteasome subunit MECL (
We have shown that preproteasomes containing LMP2 (
We have shown that in the presence of both LMP2 and LMP7, MECL is
incorporated predominantly into proteasomes that also contain both LMP2
and LMP7, which are referred to as immunoproteasomes (31). Because
ppZ.MECL is incorporated more efficiently than MECL into
Delta+/X+ proteasomes in the absence of LMP7,
we assessed whether this still occurred in the presence of both LMP2
and LMP7, which simulates the situation in which all six catalytic
subunits are expressed in the same cell. We expressed MECL.FLAG or
ppZ.MECL.FLAG with LMP2 and LMP7 in T2 cells and found that
co-precipitation of X with FLAG-tagged MECL was detectable only when
the Z propeptide was attached to MECL, demonstrating that ppZ.MECL is
more efficiently incorporated than MECL into X-containing proteasomes,
even in the presence of LMP7 (Fig. 1D). Conversely, the
absence of co-precipitating X with MECL.FLAG confirms that
incorporation of MECL and X into the same proteasome occurs
inefficiently when all of the inducible and constitutive catalytic
subunits are present. We speculate that the amount of LMP7
co-precipitating with ppZ.MECL.FLAG was not significantly reduced
because there still is relatively much more LMP7 in these proteasomes
compared with X. Co-precipitation with MECL.FLAG of relatively
more Delta than X indicates that Delta+/MECL+/LMP7+ proteasomes can
be assembled effectively when all six catalytic subunits are present.
Conversely, in the absence of LMP7,
Delta+/MECL+/X+ proteasomes are
assembled inefficiently, as demonstrated by the minimal amount of Delta
and X that co-precipitates with MECL.FLAG in that situation (Fig.
1A).
The MECL Propeptide Does Not Prevent Incorporation of Z into
Constitutive Proteasomes, but It Stabilizes
Z+/LMP2+ Preproteasomes in the
Absence of LMP7--
Because the Z propeptide enabled incorporation of
MECL ( Efficient Assembly of LMP2-containing Proteasomes Does Not Require
MECL--
The recent availability of MECL We demonstrate that differences between the MECL (1), Z (
2), and X (
5), whereas immunoproteasomes contain
three interferon-
-inducible catalytic subunits, LMP2 (
1i), MECL
(
2i), and LMP7 (
5i). We recently have demonstrated that
proteasome assembly is biased to promote immunoproteasome homogeneity
when both types of catalytic subunits are expressed in the same cell.
This cooperative assembly is due in part to differences between the
LMP7 (
5i) and X (
5) propeptides. In the current study we
demonstrate that differences between the MECL (
2i) and Z (
2)
propeptides also influence cooperative assembly. Specifically,
replacing the MECL propeptide with that of Z enables MECL incorporation
into otherwise constitutive (Delta+/X+)
proteasomes and facilitates X incorporation into otherwise
immunoproteasomes (MECL+/LMP2+). We also show,
using MECL
/
mice, that LMP2 incorporation does not
require MECL, in contrast with previous suggestions that their
incorporation is mutually codependent. These results enable us to
refine our model for cooperative proteasome assembly by determining
which combinations of inducible and constitutive subunits are favored
over others, and we propose a mechanism for how propeptides mediate
cooperative assembly.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-inducible catalytic subunits, LMP2 (
1i), MECL
(
2i), and LMP7 (
5i) (7, 8). Immunoproteasomes are thought to
possess enhanced capability for generating major histocompatibility complex class I-binding peptides with basic or
hydrophobic C termini as compared with constitutive proteasomes, which
contain three constitutively synthesized catalytic subunits, Delta
(
1), Z (
2), and X (
5) (9-14).
-type subunits,
1-
7, and each
inner ring contains seven different
-type subunits,
1-
7 (18),
at least three of which are catalytic (
1 or
1i,
2 or
2i,
and
5 or
5i). The N-terminal proteolytic active sites are on the
inner surface of the
rings, whereas the C termini of
subunits
are on the outer surface of proteasomes (19-21), enabling us to use
C-terminal "epitope tags" to immunoprecipitate and track specific
subunits because these tags do not appear to interfere with proteasome
structure or catalytic activity. For example, attaching green
fluorescent protein to the C terminus of LMP2 did not interfere with
LMP2 incorporation or proteolytic function (22).
ring, an incomplete and variable complement of unprocessed
subunits, and an assembly chaperone, "proteassemblin" (23-28).
Completion of assembly involves completion of the
ring, juxtaposition of two preproteasomes at the
ring interface,
autolysis of
subunit N-terminal propeptides, and degradation of
proteassemblin (29, 30). The
5 subunits (LMP7 and X) are
incorporated relatively late and are absent from most preproteasomes
(27). Interestingly, immunoproteasome assembly proceeds in a different
order from constitutive proteasome assembly with LMP2 (
1i) being an
early component of pre-immunoproteasomes, whereas its homologue, Delta
(
1), is a late component of constitutive preproteasomes (27).
. Originally it was thought that subunits
were incorporated according to mass action, whereas we have recently
demonstrated that immunoproteasome homogeneity is favored even in the
presence of constitutive subunits (31). Manifestations of this
cooperative assembly process include the observations that LMP2 is
required for efficient MECL incorporation and that LMP7 is incorporated
preferentially over X into proteasomes containing LMP2 and MECL (31,
32). This latter effect is dependent on differences between the LMP7
and X propeptides, as demonstrated by "propeptide switch"
experiments in which the LMP7 propeptide facilitates X incorporation
into LMP2+/MECL+ proteasomes, whereas the X
propeptide inhibits LMP7 incorporation into these same proteasomes
(33).
2i) and Z (
2) propeptides also influence cooperative proteasome
assembly. Specifically, MECL with a Z propeptide is more efficiently
incorporated into otherwise constitutive
(Delta+/X+) proteasomes, and X is more
efficiently incorporated into otherwise immunoproteasomes containing
LMP2 and MECL when MECL is expressed with a Z propeptide. We also used
cells from MECL
/
mice to establish that LMP2 does not
require MECL for efficient incorporation into proteasomes, in contrast
with MECL requiring LMP2 (31, 32). Our results enable us to refine our
model for propeptide-mediated cooperative proteasome assembly, in which certain combinations of inducible and constitutive subunits are favored
over others. Particularly inhibited are combinations of the X subunit
with any of the inducible subunits.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 and
Thr+1 (GGTACC). These restriction sites were introduced
using the Altered Sites II in vitro mutagenesis system
(Promega, Madison, WI). An internal KpnI site in the MECL
cDNA also was removed using this system. C-terminal Myc and FLAG
epitope tags were incorporated by PCR amplification, in which sequences
encoding the tags were included in the 3' primers. Human MECL cDNA
was provided by J. Monaco (University of Cincinnati College of
Medicine, Cincinnati, OH). Human Z cDNA was provided by K. Tanaka (The Tokyo Metropolitan Institute of Medical Science, Tokyo,
Japan). cDNAs encoding human Delta, LMP2, and LMP7 were expressed
in T2 cells using a stably integrated vector, pSG5 (Stratagene, La
Jolla, CA), which was co-transfected with pSV2.Neo
(Clontech, Palo Alto, CA), which confers G418
resistance. Human Delta cDNA was generated by PCR amplification of
human C1R cell cDNA using primers made from known terminal
sequences of Delta. Human LMP2 and LMP7 cDNAs have been described
previously (31). FLAG epitope tags were attached to the C termini of
Delta and LMP2 by PCR amplification. The fidelity of PCR-generated
constructs was confirmed by direct sequencing.
/
and
MECL
/
/LMP7
/
Mice, Splenic
Lymphocyte Isolation, and Immunoblotting of Mouse
Immunosubunits--
MECL-deficient mice were generated by targeted
disruption of the mouse MECL
gene.1
MECL
/
/LMP7
/
mice were generated by
crossing MECL
/
with LMP7
/
mice, the
latter obtained from H. J. Fehling (Basel Institute for
Immunology, Basel, Switzerland) (36). LMP2
/
mice were
obtained from L. Van Kaer (Vanderbilt University School of Medicine,
Nashville, TN) (37). Splenic lymphocytes were isolated by density
gradient centrifugation using underlayed Lympholyte M. Lymphocytes were stimulated with concanavalin A
(ConA)2 (5 µg/ml in R10)
for 72 h. ConA blasts were lysed with 1% digitonin, and
post-nuclear supernatants were immunoblotted, as described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2i) is co-incorporated
efficiently into proteasomes only in the presence of LMP2 (
1i), which is also an inducible subunit (31, 32). Conversely, Z (
2), the
constitutive homologue of MECL, is incorporated readily in the absence
of LMP2. We tested whether this difference is because of propeptide
differences by replacing the MECL propeptide with the Z propeptide
(ppZ.MECL). We attached C-terminal FLAG tags to MECL and ppZ.MECL and
expressed these recombinant proteins in T2 cells, which lack both LMP2
and LMP7 because of a homozygous deletion that encompasses both of
these genes. Thus, Delta is the only
1 subunit and X is the only
5 subunit expressed in these recipient cells. Analysis of
FLAG-tagged immunoprecipitated complexes from these cells demonstrated
that MECL.FLAG accumulated in an unprocessed form that was not
associated with other preproteasome subunits (pre-X, pre-Delta, or
pre-N3) and thus did not appear to be incorporated into preproteasomes
(Fig. 1A). Conversely, ppZ.MECL.FLAG was efficiently processed and incorporated into Delta+/X+ proteasomes, as demonstrated by
co-precipitation of predominantly processed MECL.FLAG with other
processed
subunits that included Delta (
1), X (
5), and N3
(
7). The lack of associated proteins with unprocessed MECL.FLAG also
was demonstrated by labeling cellular proteins with
[35S]methionine/cysteine and immunoprecipitating labeled
MECL.FLAG with anti-FLAG-Sepharose. In this experiment, no other
labeled proteins co-precipitated with MECL.FLAG (data not shown).
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Fig. 1.
A, immunoblots of anti-FLAG
immunoprecipitates of lysates from T2 cells (lane 1), T2
cells expressing MECL.FLAG (lane 2), and T2 cells expressing
ppZ.MECL.FLAG (lane 3). FLAG-tagged complexes were
immunoprecipitated from cell lysates with anti-FLAG-Sepharose and
subjected to SDS-PAGE and immunoblotting. Primary antibodies for
immunoblotting were anti-FLAG, anti-X, anti-Delta, and anti-N3.
Immunoprecipitates from 15 × 106 cells were applied
to each lane, except the N3 immunoblot that had
immunoprecipitates from 7.5 × 106 cells in each
lane. B, immunoblots of anti-FLAG
immunoprecipitates of lysates from T2 cells expressing LMP2 (lane
1), LMP2 and MECL.FLAG (lane 2), or LMP2 and
ppZ.MECL.FLAG (lane 3). Primary antibodies were anti-FLAG
and anti-LMP2. Immunoprecipitates from 5 × 106 cells
(top) or 10 × 106 cells
(bottom) were applied to each lane. It should be
noted that the pre-MECL bands in lanes 2 and 3 have different mobilities because of differences between the MECL and Z
propeptides attached to MECL in each case. C, immunoblots of
anti-FLAG immunoprecipitates of lysates from T2 cells expressing
LMP2.FLAG (lane 1), LMP2.FLAG and MECL.Myc (lane
2), or LMP2.FLAG and ppZ.MECL.Myc (lane 3). Primary
antibodies were anti-FLAG and anti-Myc. Immunoprecipitates from 15 × 106 cells were applied to each lane.
D, immunoblots of anti-FLAG immunoprecipitates of lysates
from T2 cells expressing LMP2 and LMP7 (lane 1), LMP2, LMP7,
and MECL.FLAG (lane 2), or LMP2, LMP7, and ppZ.MECL.FLAG
(lane 3). Primary antibodies were anti-FLAG, anti-X,
anti-Delta, anti-LMP2, and anti-LMP7. Immunoprecipitates from 7.5 × 106 cells were applied to each lane of the
anti-FLAG blot, 20 × 106 cells to each
lane of the anti-X and anti-Delta blots, and 15 × 106 cells to each lane of the anti-LMP2 and
anti-LMP7 blots. Each set of immunoblots in this figure and in Fig. 2
was repeated at least once, with significant differences as shown in
panels A and B being repeated at least three
times.
1i) and
MECL (
2i) accumulate in the absence of LMP7 (
5i) because of the
relatively inefficient incorporation of X (
5), which is the constitutive homologue of LMP7, into these otherwise immunoproteasomes (31). We tested whether the MECL propeptide contributed to this effect
by co-expressing MECL.FLAG or ppZ.MECL.FLAG with LMP2 in T2 cells.
Analysis of FLAG-tagged immunoprecipitated complexes from these cells
demonstrated that MECL.FLAG accumulated in an unprocessed form that
surprisingly co-precipitated a minimal amount of unprocessed LMP2 (Fig.
1B). Thus, most of the unprocessed MECL.FLAG in these cells
appeared not to be incorporated into preproteasomes. Conversely,
ppZ.MECL.FLAG was processed efficiently and co-precipitated with
processed LMP2, demonstrating increased assembly efficiency of
LMP2+/MECL+/X+ proteasomes when the
MECL propeptide is replaced by the Z propeptide. We could not directly
demonstrate increased incorporation of X into LMP2+
proteasomes because more efficient assembly of
Delta+/MECL+/X+ proteasomes also
occurred in these cells, as it did in T2 cells transfected with
ppZ.MECL.FLAG (data not shown). We speculate that the poor
incorporation of pre-MECL.FLAG into preproteasomes even in the presence
of LMP2 is because of competition with a large pool of endogenous MECL,
as suggested by Groettrup et al. (32), who found that
overexpression of MECL mRNA in T2 cells with or without LMP2 and
LMP7 does not increase the amount of MECL present in the 20 S
proteasomes. The finding that the Z propeptide, when attached to
MECL, facilitated assembly of
LMP2+/MECL+/X+ proteasomes was
confirmed by co-expressing MECL.Myc or ppZ.MECL.Myc with LMP2.FLAG in
T2 cells, where we found minimal incorporation of MECL.Myc into
complexes containing LMP2.FLAG. However, attaching the Z propeptide to
MECL.Myc significantly increased the amount of processed MECL.Myc
relative to pre-MECL.Myc that co-precipitated with LMP2.FLAG (Fig.
1C). The presence of ppZ.MECL.Myc did not reduce
significantly the accumulation of pre-LMP2.FLAG that is observed in the
absence of LMP7 because of the relatively large pool of endogenous MECL
in these cells.
2i) into otherwise constitutive
(Delta+/X+) proteasomes, we tested whether the
MECL propeptide inhibited incorporation of Z (
2) into constitutive
proteasomes by expressing Z.FLAG or ppMECL.Z.FLAG in T2 cells.
Analysis of FLAG-tagged immunoprecipitated complexes from these
cells showed that attaching the MECL propeptide to Z had a minimal
effect on Z incorporation and processing, as demonstrated by a small
increase in the amount of pre-Z.FLAG relative to the amount of
processed Z.FLAG, and had negligible effects on the amounts of
co-precipitating Delta, X, and N3 (Fig.
2A). Because the Z propeptide
facilitated assembly of
MECL+/LMP2+/X+ proteasomes in the
absence of LMP7 (Fig. 1, B and C), we also investigated whether the MECL propeptide, when attached to Z, facilitated the assembly of
Z+/LMP2+/X+ proteasomes by
expressing Z.FLAG or ppMECL.Z.FLAG with LMP2 in T2 cells. Analysis of
FLAG-tagged immunoprecipitated complexes from these cells revealed that
the MECL propeptide, when attached to Z, increased the content of
Z+/LMP2+ preproteasomes, as demonstrated by
increased pre-Z.FLAG, pre-LMP2, and proteassemblin, but it did not
facilitate the assembly of Z+/LMP2+/X+ proteasomes, as
demonstrated by similar minimal amounts of mature Z.FLAG and LMP2
(Fig. 2B). This result was confirmed by expressing Z.Myc or
ppMECL.Z.Myc with LMP2.FLAG in T2 cells, where there was a greater
amount of unprocessed ppMECL.Z.Myc in association with LMP2.FLAG as
compared with Z.Myc, demonstrating the increased steady state level of
Z+/LMP2+ preproteasomes in the absence of LMP7
when the MECL propeptide is attached to Z (Fig. 2C). The
increase in Z+/LMP2+ preproteasomes is because
of increased stability (i.e. decreased maturation and/or
degradation) of preproteasomes rather than increased formation, as we
observed no difference in the rate of incorporation of Z.Myc
versus ppMECL.Z.Myc into FLAG-tagged LMP2+
preproteasomes in pulse-chase experiments (data not shown). Finally, we
assessed whether the MECL propeptide attached to Z had an effect on Z
incorporation into LMP2+/LMP7+ proteasomes by
expressing Z.Myc or ppMECL.Z.Myc with LMP2.FLAG and LMP7 in T2 cells.
Analysis of FLAG-tagged immunoprecipitated complexes indicated that the
MECL propeptide minimally increased the incorporation of Z into these
proteasomes, as demonstrated by the slightly greater amount of Z.Myc
relative to co-precipitating LMP2.FLAG and LMP7 (Fig.
2D).
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Fig. 2.
A, immunoblots of anti-FLAG
immunoprecipitates of lysates from T2 cells (lane 1), T2
cells expressing Z.FLAG (lane 2), or ppMECL.Z.FLAG
(lane 3). FLAG-tagged complexes were immunoprecipitated from
cell lysates with anti-FLAG-Sepharose and subjected to SDS-PAGE and
immunoblotting. Primary antibodies were anti-FLAG, anti-X, anti-Delta,
and anti-N3. Immunoprecipitates from 15 × 106 cells
were applied to each lane. B, immunoblots of
anti-FLAG immunoprecipitates of lysates from T2 cells expressing LMP2
(lane 1), LMP2 and Z.FLAG (lane 2), or LMP2 and
ppMECL.Z.FLAG (lane 3). Primary antibodies were anti-FLAG,
anti-LMP2, and anti-proteassemblin. Immunoprecipitates from 15 × 106 cells were applied to each lane.
C, immunoblots of anti-FLAG immunoprecipitates of lysates
from T2 cells expressing LMP2.FLAG (lane 1), LMP2.FLAG and
Z.Myc (lane 2), or LMP2.FLAG and ppMECL.Z.Myc (lane
3). Primary antibodies were anti-FLAG and anti-Myc.
Immunoprecipitates from 15 × 106 cells were applied
to each lane. D, immunoblots of anti-FLAG
immunoprecipitates of lysates from T2 cells expressing LMP2.FLAG and
LMP7 (lane 1), LMP2.FLAG, LMP7, and Z.Myc (lane
2), or LMP2.FLAG, LMP7, and ppMECL.Z.Myc (lane 3).
Primary antibodies were anti-FLAG, anti-Myc, and anti-LMP7.
Immunoprecipitates from 8 × 106 cells were applied to
each lane.
/
mice
provided the opportunity to investigate the role of MECL in
immunoproteasome assembly using MECL-deficient cells. We derived
ConA-stimulated splenic T-cell lymphoblasts (ConA blasts) from mice
deficient in each of the immunosubunits and immunoblotted cell lysates
to assess the steady state levels of each immunosubunit and its
precursors (Fig. 3A).
Wild-type ConA blasts express predominantly immunosubunits, although
basal expression of constitutive homologues in these cells can
compensate when an immunosubunit is absent (31). Absence of MECL did
not affect significantly the level of LMP2, and it did not result in an
accumulation of precursor LMP2, unlike what is observed in the absence
of LMP7, where LMP2+/MECL+ preproteasomes
accumulate because of inefficient incorporation of X (31). The absence
of MECL also did not affect the levels of LMP7 and pre-LMP7, similar to
what is observed in the absence of LMP2, indicating that LMP7 can be
incorporated efficiently with any combination of
1 and
2
subunits. We crossed MECL
/
with LMP7
/
mice to generate mice deficient in both of these immunosubunits, from
which we derived ConA blasts and immunoblotted cell lysates for LMP2
(Fig. 3B). Absence of LMP7 resulted in accumulation of pre-LMP2, whether MECL was present or absent, although the absence of
MECL appeared to decrease the ratio of pre-LMP2 to mature LMP2. This
result suggests that although the presence of MECL may reduce the
incorporation of X into otherwise immunoproteasomes, it is not fully
responsible for this effect. Conclusions based on steady state subunit
levels were supported by pulse-chase metabolic labeling of ConA blasts
from wild-type, MECL-1
/
,
MECL-1
/
/LMP7
/
, and
LMP7
/
mice. Cells were pulsed with
[35S]methionine/cysteine for 45 min and then chased for 0 or 4 h, which under normal circumstances readily labels
preproteasome subunits with the 45-min pulse and then chases most of
the label out of the preproteasomes and into the 20 S proteasomes with
the 4-h chase (29). We immunoprecipitated preproteasomes from
pulse-chase-labeled cell lysates using the preproteasome-specific
anti-C8 antibody, and we visualized labeled preproteasome subunits by
two-dimensional gel electrophoresis and autoradiography (Fig.
4). After the 4-h chase, very little
label remained in the preproteasomes in either wild-type or
MECL-deficient cells, whereas most of the label was not chased out of
the preproteasomes in either LMP7-deficient cells or cells deficient in
both LMP7 and MECL. Thus, the inefficient assembly of proteasomes
containing LMP2 that is observed in the absence of LMP7 is largely
independent of the presence of MECL.
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Fig. 3.
A, immunoblots of lysates from ConA
blasts derived from wild-type B6 mice (lane 1),
MECL /
mice (lane 2), LMP7
/
mice (lane 3), and LMP2
/
mice (lane
4). Primary antibodies were anti-MECL, anti-LMP7, and anti-LMP2.
Lysates from 5 × 106 cells were applied to each
lane. B, anti-LMP2 immunoblot of lysates from
ConA blasts derived from wild-type B6 mice (lane 1),
LMP2
/
mice (lane 2), LMP7
/
mice (lane 3), MECL
/
mice (lane
4), and MECL
/
/LMP7
/
mice
(lane 5). Lysates from 5 × 106 cells were
applied to each lane.
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Fig. 4.
Two-dimensional NEPHGE-PAGE of radiolabeled
preproteasomes analyzed by fluorography. ConA blasts derived from
wild-type B6 mice (panels A and B),
MECL /
mice (panels C and D),
MECL
/
/LMP7
/
mice (panels E
and F), and LMP7
/
mice (panels G
and H) were metabolically labeled with
[35S]methionine/cysteine for 45 min. One-half of each
sample was harvested at that point (panels A, C,
E, and G), while the other half was chased with
cold methionine for 4 h (panels B, D,
F, and H). Lysates from 5 × 106
cells for each panel were immunoprecipitated with anti-mouse
C8 that immunoprecipitates preproteasomes but not fully assembled 20 S
proteasomes (27).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2i) and Z
(
2) propeptides influence proteasome assembly at two levels,
2
subunit incorporation into preproteasomes and the assembly of
catalytically active 20 S proteasomes. The influence on subunit incorporation is demonstrated by the ability of the Z propeptide to
enhance MECL incorporation into Delta+/X+
proteasomes (Fig. 1A), whereas the effect on 20 S proteasome assembly is demonstrated by the enhanced assembly of
LMP2+/MECL+ proteasomes in the absence of LMP7
(Fig. 1B). Taken together with our previous work, our
present findings allow us to refine our model for cooperative assembly
and propose a mechanism for propeptide involvement. Fig.
5 shows eight pathways that lead to
combinations of catalytic subunits in a proteasome
ring and indicates which pathways are inhibited by cooperative assembly. Pathway
1 leads to homogeneous immunoproteasomes and is favored over pathway 2, in which X substitutes for LMP7, as demonstrated by the accumulation of
MECL+/LMP2+ preproteasomes in cells expressing
LMP2 but not LMP7 (Figs. 1B, 1C, 3A,
and 4) (31). The influence of propeptides is demonstrated by the
observation that pathway 2 occurs more efficiently when the MECL
propeptide is replaced by the Z propeptide (Fig. 1, B and
C). Pathway 3 that leads to proteasomes containing the
constitutive subunit Z in combination with LMP2 and LMP7 is not
inhibited by cooperative assembly, as demonstrated both by efficient
incorporation of Z into proteasomes containing LMP2.FLAG and LMP7 (Fig.
2D) and by normal levels of LMP2 and LMP7 in ConA blasts
from MECL
/
mice where Z is the only available
2
subunit (Fig. 3A). Pathway 3 is favored over pathway 4, in
which X substitutes for LMP7, as demonstrated by accumulation of
Z+/LMP2+ preproteasomes both in T2 cells
expressing LMP2.FLAG and Z.Myc (Fig. 2C) and in ConA blasts
from MECL
/
/LMP7
/
mice (Fig.
3B). Pathway 5 leads to proteasomes containing the constitutive subunit Delta in combination with MECL and LMP7. This
pathway is inhibited to some degree by cooperative assembly, as
demonstrated by accumulation of MECL+ preproteasomes in
ConA blasts from LMP2
/
mice (Fig. 3A).
Pathway 5 does occur when all six subunits are present, as
demonstrated by the presence of Delta but not X in MECL+
proteasomes when LMP7 is present (Fig. 1D); also
demonstrated is the preference for pathway 5 over pathway 6, in which X
substitutes for LMP7. The influence of propeptides on
cooperative assembly again is demonstrated by the observation that
pathway 6, formed through the incorporation of MECL into
Delta+/X+ proteasomes, occurs more efficiently
when the MECL propeptide is replaced by the Z propeptide (Fig.
1A). Pathway 7 that leads to proteasomes containing the
inducible subunit LMP7 in combination with Z and Delta is not inhibited
by cooperative assembly, as demonstrated by normal levels of LMP7 in
ConA blasts from either MECL
/
or LMP2
/
mice (Fig. 3A). Finally, pathway 8 that leads to homogeneous constitutive proteasomes is favored by cooperative assembly, as demonstrated by efficient incorporation of Delta and X into
Z+ proteasomes in the absence of LMP2 and LMP7 (Fig.
2A).
View larger version (24K):
[in a new window]
Fig. 5.
Alternative pathways for incorporation of
catalytic subunits into subunit rings. Subunits are
identified by the following symbols: M, MECL; 2,
LMP2; 7, LMP7; and
, Delta. Inducible subunits
are yellow, and constitutive subunits are blue.
1 subunits are squares,
2 subunits are
circles, and
5 subunits are triangles.
We speculate that propeptides influence cooperative assembly via
differential propeptide-protein interactions and propose the following
hypothetical mechanism based on our data. MECL (2i) normally
requires LMP2 (
1i) for efficient co-incorporation into preproteasomes, which occurs relatively early during assembly, noting
that LMP2 is incorporated earlier than its homologue Delta (
1).
Conversely, Z (
2) appears to be incorporated into preproteasomes before Delta and thus is incorporated without an adjoining
1 subunit. Replacing the MECL propeptide with the Z propeptide obviates the need of MECL for LMP2, and we suggest that the Z propeptide enables
MECL to be incorporated without an adjoining
1 subunit. We further
suggest that the X propeptide may inhibit X incorporation into
preproteasomes that already contain a
1 subunit. Thus, X has a more
difficult time being incorporated into LMP2+ proteasomes
than Delta+ proteasomes because LMP2 is incorporated
earlier than Delta. Thus, if the Z propeptide enhances incorporation of
MECL into preproteasomes without a
1 subunit, it could enhance X
incorporation. This mechanism could explain not only the enhanced
assembly of Delta+/MECL+/X+
proteasomes but also the increased incorporation of X into
LMP2+/MECL+ proteasomes where the incorporation
of LMP2 and MECL are no longer linked because of the presence of the Z
propeptide. To identify the propeptide-protein interactions that
mediate these effects, we currently are determining functionally
important propeptide residues. Analysis of the amino acid sequences of
the human MECL and Z propeptides reveals a high degree of homology
(Fig. 6A), yet there are a
number of non-conservative differences that may be functionally
important, particularly residues
17 to
12 of the Z propeptide,
where the sequence ADFAKR compares to RVLP at
15 to
12 of the MECL
propeptide. Fig. 6B compares the sequence of the human Z
propeptide with
2 propeptides from other eukaryotes. Many of the
residues that are conserved among these
2 propeptides are the same
residues that are identical between the human Z and MECL propeptides.
Conservation of these residues in non-vertebrates that lack
immunosubunits suggests that they may be important for propeptide
functions other than cooperative assembly. Interestingly, residues
17
to
14 (ADFA) of the mammalian propeptides represent an insertion as
compared with non-vertebrates. The uniqueness of this segment to
mammalian propeptides and its difference between the Z and MECL
propeptides suggests that these residues may play a role in cooperative
assembly.
|
In summary, differences between the MECL and Z propeptides influence
cooperative proteasome assembly, with the Z propeptide able to overcome
inhibition of pathways that lead to co-incorporation of MECL and X. It
is unclear why the assembly of certain combinations of catalytic
subunits is inhibited by cooperative assembly, but understanding the
mechanisms of cooperativity will provide tools to investigate this
question and advance our understanding of proteasome function in cell
biology and immune responses.
![]() |
ACKNOWLEDGEMENT |
---|
We thank David B. Ginsburg for expert technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by an Arthritis Investigator Award (to T. A. G.), National Institutes of Health Grant AR02013 (to R. A. C.), and the Children's Hospital Research Foundation of Cincinnati Children's Hospital Medical Center.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.
¶ To whom correspondence should be addressed: 3333 Burnet Ave., Cincinnati, OH 45229. Tel.: 513-636-3338; Fax: 513-636-5331; E-mail: grift0@cchmc.org.
Published, JBC Papers in Press, November 26, 2002, DOI 10.1074/jbc.M209292200
1 L. Elenich and J. J. Monaco, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are: ConA, concanavalin A; ConA blasts, concanavalin A-stimulated splenic T-cell lymphoblasts; NEPHGE, non-equilibrium pH gradient electrophoresis.
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