 |
Introduction |
Proteasomes are multisubunit multicatalytic proteases
that are responsible for the majority of nonlysosomal
protein degradation within eukaryotic cells (1), and have a
central role in the generation of peptides presented by
MHC class I molecules (2). The 20S catalytic core (20S
proteasome) is composed of 28 subunits assembled in four
stacked seven-membered rings (3). The outer rings contain
seven different noncatalytic
-type subunits, and the inner
rings contain seven different
-type subunits, three of
which are catalytic (delta, X, and Z; reference 4) (alternative nomenclature for vertebrate proteasome subunits [3]:
iota,
1; C3,
2; C9,
3; C6,
4; zeta,
5; C2,
6; C8,
7; delta, Y or
1; LMP2,
1i; Z,
2; MECL,
2i; C10,
3; C7,
4; X, MB1 or
5; LMP7,
5i; C5,
6; N3, beta
or
7). In addition to seven constitutively synthesized
subunits, vertebrates have three IFN-
-inducible
subunits (LMP2, LMP7, and MECL), the former two being
encoded in the MHC (5). All three inducible subunits
have removable presequences and are catalytically active
(7). Each inducible subunit is homologous with a constitutive catalytic subunit (LMP2/delta, LMP7/X, and MECL/Z),
and can replace its homologue during proteasome assembly
(7, 12). The inducible subunits appear to be responsible for altered peptidase specificities in IFN-
-treated cells
(13), transfected cells (16), and cells from LMP7
/
and LMP2
/
mice (19, 20). Presentation of certain antigens is diminished in LMP2
/
and LMP7
/
mice (20,
21), and in the case of LMP7
/
mice, MHC class I expression is reduced (21). These results support a role for inducible subunits in enhancing proteasomal generation of
MHC class I-binding peptides.
The assembly of 20S proteasomes and the mechanism by
which inducible subunits replace constitutive homologues
are poorly understood. We have recently characterized
proteasome assembly in mouse cells expressing both inducible and constitutive catalytic subunits using an antibody to
an
subunit, anti-C8, that immunoprecipitates only 12-16S preproteasomes (22). These catalytically inactive precursor complexes (~300 kD) contain all seven
subunits
and some unprocessed
subunits. They appear to assemble
in two stages, with certain unprocessed
subunits (pre-Z,
pre-LMP2, pre-MECL, C10, and C7) being incorporated
before others (pre-C5, pre-delta, and pre-LMP7). Maturation of preproteasomes to 20S proteasomes (~700 kD) involves the juxtaposition of two preproteasomes at the
ring interface (3), with
subunit presequences being removed coincident with completion of assembly (23, 24). It is unknown whether the incorporation of inducible subunits and their homologues into proteasomes depends only
on relative expression levels, or whether certain proteasome forms are assembled preferentially.
 |
Materials and Methods |
Episomal Expression Vectors.
pCEP4 (ampicillinr, hygromycinr) and pREP9 (ampicillinr, neomycinr) were purchased from
Invitrogen (Carlsbad, CA). pCEP9 (ampicillinr, neomycinr) was
constructed from three DNA fragments: SalI-XbaI (1,377 to 2) from
pREP9, XbaI-BamHI (1 to 405) from pCEP4, and BamHI-SalI (405 to 1,315) from pCEP4. pCEP9 is similar to pCEP4 except
the hygromycin resistance gene replaces the neomycin resistance
gene. pCEP9.LMP2 was constructed by inserting at HindIII-
BamHI a full-length human LMP2 cDNA, obtained from H.O.
McDevitt (Stanford University School of Medicine, Stanford,
CA) (25). pCEP4.LMP7 was constructed by inserting at KpnI-
BamHI a full-length human LMP7 cDNA, obtained from T. Spies (Fred Hutchinson Cancer Research Center, Seattle, WA)
(10). pCEP4.LMP7E1 was constructed using synthetic oligonucleotides to change only the presequence of LMP7E2. The promoter
and translation control sequences upstream of the start codon were
unchanged; thus, transcription and translation efficiencies were
expected to be similar to LMP7E2. pCEP4.LMP7(T1A),
pCEP4.LMP7(K33A), pCEP9.LMP2(T1A), and pCEP9.LMP2
(K33A) were constructed by site-directed mutagenesis using the
Altered sites® II in vitro mutagenesis system (Promega, Madison,
WI) or the QuickchangeTM site-directed mutagenesis kit (Stratagene Corp., La Jolla, CA).
Antibodies.
MCP21 is a mouse monoclonal antibody that recognizes human C3 and immunoprecipitates C3-containing 20S
proteasomes and 12-16S preproteasomes (26). MCP21 ascites
fluid was obtained from K.B. Hendil and the hybridoma expressing MCP21 was obtained from the European Collection of Animal Cell Cultures (Salisbury, Wiltshire, UK). Polyclonal antisera
recognizing LMP2, LMP7, MECL, delta, Z, C8, C9, or TAP2
were from rabbits immunized with recombinant mouse subunits
(or rat for anti-C9). Rabbit polyclonal antisera raised against human LMP2 and LMP7 were obtained from H.O. McDevitt (Stanford University School of Medicine, Stanford, CA) (25). Anti-X (P93250), obtained from K.B. Hendil (August Krogh Institute, University of Copenhagen, Copenhagen, Denmark), is a
rabbit polyclonal antiserum raised against human X (18).
Cell Culture.
T2 cells, obtained from P. Cresswell (Howard
Hughes Medical Institute, Yale University School of Medicine,
New Haven, CT) (27), were transfected with episomal expression vectors by electroporation (250 V and 500 µFd) in serum-free RPMI at 2.5 µg DNA/5 × 106 cells. Transfected cells were
grown in RPMI with 10% FCS (R10) for 48 h before the addition of selection antibiotic(s). Selected cells were maintained in
RPMI with 5% calf serum with either G418 at 0.8 mg activity/
ml (GIBCO BRL, Gaithersburg, MD) for pCEP9 and/or hygromycin at 360 U/ml (Calbiochem Corp., La Jolla, CA) for
pCEP4. Con A-stimulated T cell blasts were prepared by growing mouse spleen lymphocytes, isolated using lympholyte M, in
R10 with Con A (5 µg/ml) for 72 h.
Immunoprecipitation and Immunoblotting.
T2 cells were lysed
with 1% NP-40 in 20 mM Tris, pH 7.6, 10 mM EDTA, and 100 mM NaCl. Proteasomes were immunoprecipitated from postnuclear supernatants with MCP21 (1 µg/106 cells) and protein
G-Sepharose (250 µg/106 cells). Immunoprecipitates were boiled
in 2 × SDS sample buffer, subjected to 12.5% SDS-PAGE, and
electroblotted onto polyvinylidene difluoride paper at 500 mA
(milliamps) for 90 min. Specific proteins were detected using
antisera or MCP21 as primary antibodies, alkaline phosphatase
conjugated goat anti-rabbit or goat anti-mouse as secondary antibodies, and 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium for color development. Mouse spleen Con A-stimulated T cell blasts were lysed with 1% digitonin and postnuclear
supernatants were boiled in 6 × SDS sample buffer and immunoblotted as above.
Sucrose Gradient Centrifugation.
NP-40 cell lysates (1 ml) were
layered onto 10-ml sucrose gradients (15-35% in NP-40 lysis
buffer) and ultracentrifuged in a rotor (SW40 Ti; Beckman, Fullerton, CA) at 40,000 rpm for 20 h at 4°C. Fractions (1 ml × 11)
were collected from the bottom of the tubes.
Metabolic Labeling, Immunoprecipitation, and Two-dimensional Gel
Electrophoresis.
Con A-stimulated T cell blasts derived from
mouse spleens (~20 × 106 cells) were labeled with 0.5 mCi
[35S]methionine/cysteine for 45 min. Half the cells were lysed
with 0.5% NP-40 and the rest were chased for 8 h in R10 containing 4 mM cold methionine and then lysed. 12-16S preproteasomes were immunoprecipitated with anti-C8, whereas 20S proteasomes and a small subset of 12-16S preproteasomes were
immunoprecipitated with anti-C9 (22). Immunoprecipitates were
resuspended in nonequilibrium pH gradient electrophoresis
(NEPHGE)1 sample buffer and subjected to two-dimensional
NEPHGE-PAGE and autoradiography (22).
 |
Results |
LMP7 Mediates Efficient LMP2 Processing in Transfected T2
Cells.
T2 is a human lymphoblastoid cell line with a single copy of chromosome 6, which has a deletion in the
MHC class II region that includes the genes for LMP2 and
LMP7 (27, 28). We observed that proteasomes from T2
cells transfected with LMP2 (T2.LMP2) contained primarily unprocessed LMP2 (pre-LMP2) (Fig. 1). The relative
amount of pre-LMP2 decreased when cells were overgrown; however, we always detected more of the precursor than the mature form (data not shown), suggesting that
processing of LMP2 was inefficient in these cells. A similar
observation has been made using .174 lymphoblasts (which
also lack LMP2 and LMP7) transfected with LMP2 (16).
To determine whether LMP7 could mediate efficient processing of LMP2, T2 cells were transfected with both
LMP2 and LMP7 (T2.LMP2/LMP7). Proteasomes from
these double transfectants contained predominantly processed LMP2 (Fig. 1), indicating that LMP7 can indeed enhance the efficiency of LMP2 processing. In contrast, there
was no effect of LMP2 on the efficiency of LMP7 processing, as proteasomes from both double transfectants as well as T2 cells transfected with LMP7 alone (T2.LMP7) contained primarily processed LMP7. Immunoblotting with
polyclonal antisera raised against human LMP2 and LMP7
produced identical results but greater background staining
(data not shown). Consequently, all of the anti-LMP2 and
anti-LMP7 immunoblots presented in this report use antisera raised against mouse subunits. Recently, Groettrup et al. did not observe that efficient LMP2 processing required
LMP7 in transfected T2 cells (29). Their results may differ
from ours because they used an expression vector (pSG5)
that leads to significant overexpression of LMP2, and they
used mouse LMP2 and LMP7 in this human cell line.

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Fig. 1.
Effect of LMP7 on processing of LMP2 in transfected T2 cells.
Proteasomes were immunoprecipitated with MCP21 from lysates (postnuclear supernatants) of T2 cells transfected with LMP2 (2), LMP2 and
LMP7 (2 + 7 ), LMP2 and LMP7E1 (2 + 7E1), LMP7 (7), and LMP7E1
(7E1). Specific subunits were visualized by immunoblotting after SDS-PAGE. Anti-LMP2 and anti-LMP7 antisera were raised against mouse
subunits and cross-react with human subunits. Immunoprecipitates from
5 × 106 cells were loaded per lane. The C3 immunoblot demonstrates the relative amounts of proteasomes in each sample.
|
|
LMP7E1 Is Inefficiently Incorporated into Proteasomes and
Fails to Mediate Efficient LMP2 Processing in Transfected T2
Cells.
There are two forms of human LMP7 (E1 and E2)
which result from alternative first exon usage (10). These
two forms have different amino acid sequences only in
their presequences (NH2 terminus to residue
24), with
the mature proteins being identical. LMP7E2 is the predominant form expressed in tissues and cell lines (10), and
this form of LMP7 is used throughout our work except where noted. It has been shown that LMP7E1 is inefficiently incorporated into proteasomes in transfected HeLa
cells (24). We obtained a similar result using transfected T2 cells
(T2.LMP7E1; Fig. 1). Interestingly, LMP2 was inefficiently
processed in T2.LMP2/LMP7E1 cells where LMP7E1 was
incorporated inefficiently. These results indicate that the
nature of the LMP7 presequence is important for LMP7
incorporation into proteasomes, and LMP7 incorporation is necessary for efficient LMP2 processing. We also found
low steady state levels of free LMP7E1 in transfected T2
cells (data not shown), suggesting that LMP7E1 is relatively
unstable when not incorporated into proteasomes.
LMP7 Catalytic Activity Is Not Required for Efficient LMP2
Processing in Transfected T2 Cells.
To determine whether catalytic activity of LMP7 is required for its effect on LMP2
processing, catalytically inactive forms of LMP7 were produced by mutating active site residues threonine 1 or lysine
33 to alanine (T1A or K33A). Neither of these point mutations diminished incorporation of LMP7 into proteasomes,
nor did they reduce the ability of LMP7 to enhance LMP2
processing (Fig. 2). Both mutants failed to undergo complete autocatalytic processing (30), resulting in higher
molecular weight partially processed forms, as seen with active site mutations of other catalytic
subunits (31, 32).
We also produced catalytically inactive forms of LMP2
(T1A or K33A), which were unprocessed in the absence of
LMP7 (like wild-type LMP2) and partially processed in the
presence of either LMP7 or its catalytically inactive forms.
LMP2 inactivity did not affect LMP7 processing (data not
shown). Taken together, these results demonstrate that catalytic activity of LMP7 is not required for its effect on
LMP2 processing, nor is LMP2 catalytic activity required
for this effect.

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Fig. 2.
Effect of LMP7 active site mutations on LMP2 processing.
Proteasomes were immunoprecipitated with MCP21 from lysates of T2 cells transfected with LMP2 and LMP7 (2 + 7), LMP2 and LMP7(T1A) [2 + 7(T1A)], and LMP2 and LMP7(K33A) [2 + 7(K33A)]. Specific subunits were visualized by immunoblotting after SDS-PAGE. Immunoprecipitates from 107 cells were loaded per lane. ppLMP7, partially processed LMP7. Partial loss of the pre-LMP7 band in lane 2 + 7 is due to
poor transfer as a result of an air bubble.
|
|
Pre-LMP2 Accumulates in Preproteasomes in the Absence of
LMP7 in Transfected T2 Cells.
Removal of
subunit presequences is associated with maturation of 20S proteasomes
(23, 24). Therefore, we hypothesized that pre-LMP2,
which accumulates in T2.LMP2 cells, remains in preproteasomes. To test this, cell extracts were fractionated on sucrose gradients and C3-containing proteasomes (both 20S
proteasomes and 12-16S preproteasomes) were immunoprecipitated from each fraction and analyzed by immunoblotting for specific subunits (Fig. 3). Indeed, pre-LMP2
was found exclusively in lower mol wt fractions 4 and 5, comigrating with a portion of C3, consistent with
subunit-containing preproteasomes. The small amount of processed LMP2 in these cells (fraction 2) comigrated with
other processed
subunits (delta and X), as well as the majority of C3, consistent with 20S proteasomes. These results
agree with previous demonstrations of pre-LMP2 in preproteasomes from transfected T2 cells overexpressing mouse LMP2 (23, 32). Of note, there was no detectable pre-delta or pre-X in the pre-LMP2-containing fractions. In contrast, when LMP7 was coexpressed with LMP2 (T2.LMP2/
LMP7), processed LMP2 predominated and comigrated
with processed LMP7 and the bulk of C3, consistent with
20S proteasomes (Fig. 3). These results demonstrate that
preproteasomes containing pre-LMP2 accumulate in the
absence of LMP7, despite the presence of its homologue,
X. In contrast, pre-LMP2-containing preproteasomes do
not accumulate in the presence of LMP7, suggesting a
strong preference for the subsequent incorporation of LMP7
rather than X into these assembling proteasomes. It is important to note that LMP7 can be incorporated into 20S
proteasomes in the absence of LMP2 (Fig. 1 and data not
shown), suggesting that preproteasomes lacking pre-LMP2
do not have a strong preference for the incorporation of X
or LMP7.

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Fig. 3.
Sucrose gradient fractionation of proteasomes from transfected
T2 cells. Lysates of 4 × 107 cells were separated on sucrose gradients, and
then mature and precursor proteasomes were immunoprecipitated from
individual fractions with MCP21. Specific subunits were visualized by
immunoblotting after SDS-PAGE, with one-quarter of each immunoprecipitation loaded per lane. Fractions 1-6 out of 11 are shown, with fraction 1 representing the bottom of the gradient.
|
|
Pre-LMP2 and Pre-MECL Accumulate in Spleen Cells from
LMP7
/
Mice; The Level of MECL Is Reduced in Spleen
Cells from LMP2
/
Mice.
Since T2 cells lack a large portion of the MHC class II region (27), they are deficient in
not only LMP2 and LMP7, but also other genes involved
in antigen processing (TAP1 and TAP2) and potentially
other as yet undiscovered genes. Thus, we were interested in determining whether LMP7 is required for efficient
LMP2 processing in other model systems, such as mice
with targeted deletions of LMP2 or LMP7. Indeed, consistent results were obtained using Con A-stimulated T cell
blasts from spleens of B10 (wild type), LMP7
/
, and
LMP2
/
mice (Fig. 4). Immunoblots reflecting steady-state subunit levels demonstrated that all of the detectable
LMP2 in wild-type cells was processed, whereas the majority of LMP2 in cells from LMP7
/
mice was unprocessed.
Comparable to T2 transfectants, LMP2 deficiency had no
effect on LMP7 processing. Interestingly, using an antibody against mouse MECL, we found that the level of processed
MECL was dramatically reduced in the absence of either
LMP7 or LMP2. Furthermore, unprocessed MECL accumulated in the absence of LMP7, similar to the accumulation of unprocessed LMP2 in these cells, indicating that absence of LMP7 results in inefficient processing of not only
LMP2 but also MECL. Groettrup et al. have also noted that MECL incorporation into proteasomes is enhanced by
overexpression of mouse LMP2 in transfected T2 cells, and
LMP2 incorporation is enhanced by overexpression of
MECL in transfected mouse B8 fibroblasts (29). The reduced level of MECL in cells from LMP2
/
mice is possibly a consequence of increased degradation of free MECL
secondary to its inefficient incorporation into proteasomes in the absence of LMP2. Notably, reduced levels of each
processed inducible subunit correlated with increased levels
of each respective constitutive homologue. For example,
the amount of delta was increased not only in the absence
of LMP2 but also when LMP2 was processed inefficiently
in cells from LMP7
/
mice. Similarly, the amount of Z
was increased when processed MECL was reduced due to
lack of either LMP7 or LMP2. The amount of X was increased only when LMP7 was absent.

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Fig. 4.
Relative levels of proteasome subunits in spleen cells from
B10 (wild type), LMP7 / , and LMP2 / mice. Lysates of mouse spleen
Con A-stimulated T cell blasts (1.5 × 106/lane) were subjected to SDS-PAGE and specific proteasome subunits were visualized by immunoblotting. The C9 and TAP2 immunoblots demonstrate the relative amounts
of proteasomes and total protein present in each sample.
|
|
Proteasomes Are Assembled Inefficiently in Spleen Cells from
LMP7
/
and LMP2
/
Mice.
To confirm the presence
of unprocessed LMP2 and unprocessed MECL in preproteasomes from LMP7
/
mice, and to assess kinetics of
proteasome assembly in both LMP7
/
and LMP2
/
mice, Con A-stimulated T cell blasts derived from spleens
of NIH Black Swiss (wild type), LMP7
/
, and LMP2
/
mice were subjected to metabolic pulse-chase labeling (Fig.
5). 12-16S preproteasomes and 20S proteasomes were analyzed on two-dimensional gels after immunoprecipitation
with anti-C8 or anti-C9, respectively (22). Several salient
observations can be made from this experiment. First,
overall proteasome assembly in cells from LMP7
/
mice
was inefficient. Despite an apparent increase in the synthesis of proteasome subunits in these cells (45 min pulse, anti-C8), the percentage of radioactive material chasing into
20S proteasomes (45 min pulse + 8 h chase, anti-C9) was
small as compared to wild type. Second, preproteasomes
synthesized in cells from LMP7
/
mice appeared to be
longer lived than wild type (45 min pulse + 8 h chase,
anti-C8). Third, preproteasomes that accumulated in these
cells contained significant amounts of pre-LMP2 (spot p2)
and pre-MECL (spot pM) (45 min pulse + 8 h chase, anti-C8), yet very little of these particular subunits were processed and chased into 20S proteasomes (spots 2 and M) (45 min pulse + 8 h chase, anti-C9), indicating inefficient
completion of assembly of preproteosomes containing pre-LMP2 and pre-MECL in the absence of LMP7, consistent
with immunoblotting results (Fig. 4). The increased synthesis
of proteasome subunits and relatively inefficient assembly of
20S proteasomes in cells from LMP7
/
mice has been observed in three separate experiments (data not shown).
Fourth, preproteasomes in cells from LMP2
/
mice contained a small amount of pre-MECL (spot pM) (45 min
pulse), and were longer lived than wild type (45 min pulse + 8 h chase, anti-C8). However, MECL did not chase into
20S proteasomes in these cells (spot M) (45 min pulse + 8 h
chase, anti-C9), whereas processed Z, which is the dark
spot just to the left of the MECL position, is incorporated
into 20S proteasomes. This indicates that MECL, unlike its
homologue Z, is inefficiently incorporated into proteasomes in the absence of LMP2, consistent with immunoblotting results (Fig. 4) and recent studies of Groettrup et
al. (29).

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Fig. 5.
Pulse-chase labeling of proteasomes synthesized in spleen cells from NIH Black Swiss (wild type), LMP7 / , and LMP2 / mice. Equal numbers of mouse spleen Con A-stimulated T cell blasts were labeled with [35S]methionine/cysteine for 45 min and then harvested (45 minute pulse) or chased
for 8 h (45 minute pulse + 8 hour chase). At the end of each incubation, cells were lysed and proteasomes were immunoprecipitated from half of each lysate
with anti-C8, which recognizes only 12-16S preproteasomes (anti-C8), and from the other half of each lysate with anti-C9, which recognizes 20S proteasomes and a small subset of 12-16S preproteasomes (anti-C9) (22). (Anti-C9 immunoprecipitates after a 45-min pulse demonstrated little radioactivity in
20S proteasomes and are not shown.) Proteasome subunits were separated by two dimensional NEPHGE-PAGE and visualized by autoradiography.
Spots corresponding to inducible subunits and their precursors, as identified previously (22), are labeled (p2, pre-LMP2; 2, LMP2; p7, pre-LMP7; 7,
LMP7; pM, pre-MECL; M, MECL). Note that pre-LMP7 is partially obscured by a larger spot (iota) in NIH Black Swiss preproteasomes. LMP7, LMP2,
and their precursors have slightly different isoelectric points in NIH Black Swiss mice due to known polymorphisms (34). Immunoprecipitates from 5 × 106 cells were loaded on each gel.
|
|
 |
Discussion |
Based on our results, we propose a cooperative model
for immunoproteasome assembly (Fig. 6). When both
IFN-
-inducible and constitutive catalytic
subunits are
synthesized, either pre-LMP2 and pre-MECL are cooperatively incorporated early into preproteasomes or, alternatively, pre-Z is incorporated early. The relative levels of
pre-LMP2 and pre-MECL compared to pre-Z may determine which major pathway is favored (33), although differences in affinity of each of these precursor subunits for
preproteasomes could also be important. Nevertheless, preproteasomes containing pre-LMP2 and pre-MECL favor
the incorporation of LMP7, since these preproteasomes do
not efficiently mature when LMP7 is absent and its homologue X is present. This cooperative incorporation of LMP7
into preproteasomes containing pre-LMP2 and pre-MECL
leads to 20S immunoproteasomes containing all three IFN-
-inducible subunits. Cooperative incorporation of constitutive catalytic subunits may also occur, with preproteasomes containing pre-Z favoring the subsequent incorporation of
delta and X. Exclusion of X from the immunoproteasome
pathway may increase its availability for incorporation into
constitutive proteasomes. Evidence supporting this comes
from Gaczynska et al. who have shown that overexpression
of X increases the content of delta and decreases the content of LMP2 in proteasomes (18). Despite cooperativity,
some mixed proteasomes may form, since LMP7 can be incorporated into proteasomes in the absence of LMP2.

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Fig. 6.
Cooperative model for proteasome assembly. One major
pathway leads to immunoproteasomes containing all three IFN- -inducible catalytic subunits (LMP2, LMP7, and MECL). Another major
pathway leads to constitutive proteasomes containing all three constitutive catalytic subunits (delta [ ], X, and Z). Minor pathways lead to mixed
proteasomes containing assortments of both inducible and constitutive
catalytic subunits. Appendages represent removable presequences. Subunit positions are based on the structure of the yeast 20S proteasome and
assume that inducible subunits occupy the same sites as their constitutive
homologues (3).
|
|
Cooperative incorporation of inducible subunits may
serve several important functions. First, it favors the assembly of homogeneous "immunoproteasomes", which would
be important if certain antigen-processing functions of inducible subunits occur in concert. This is supported by the
observation that LMP7
/
mice, which have reduced levels of all three processed inducible subunits, have a more
profound phenotype (reduced MHC class I expression)
than LMP2
/
mice, which have reduced levels of only
LMP2 and MECL. Second, it discourages assembly of heterogeneous "mixed" proteasomes containing mass-action
random assortments of inducible and constitutive catalytic subunits. This may be important in the antiviral response
where IFN-
upregulation of inducible subunits occurs
without downregulation of constitutive subunits (7, 12),
which could result in the assembly of mixed proteasomes
that generate peptides not normally produced during T cell
development and lead to autoimmune reactivity. Third, it
increases the likelihood that at least some homogeneous "constitutive" proteasomes are assembled in lymphoid tissues where inducible subunits predominate. This would be
important if certain vital housekeeping functions of constitutive proteasomes are not served by immunoproteasomes.
In summary, we demonstrate a mechanism at the protein
level which favors the assembly of immunoproteasomes
containing all three IFN-
-inducible catalytic
subunits in
the same 20S complex. This mechanism depends on the
nature of the inducible subunits, including presequences,
but not on their catalytic activity. Incorporation of these
subunits into proteasomes does not appear to be simply a
function of relative levels of expression. Our cooperative
model for proteasome assembly suggests an important role
for ensuring the assembly of either homogeneous immunoproteasomes and/or homogeneous constitutive proteasomes in cells and tissues expressing both inducible and
constitutive catalytic subunits to varying degrees.
Address correspondence to Dr. Robert A. Colbert, William S. Rowe Division of Rheumatology, Children's
Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229. Phone: 513-636-4934; Fax: 513-636-5831; E-mail: bob.colbert{at}chmcc.org
We thank Klavs B. Hendil for generous gifts of MCP21 ascites fluid and anti-human X antiserum, and
David B. Ginsburg for technical assistance.
This work was supported in part by a Trustee Grant from the Children's Hospital Research Foundation of
Cincinnati, the Schmidlapp Foundation, and the National Institutes of Health. T.A. Griffin was supported
by an Arthritis Foundation Postdoctoral Fellowship. D. Nandi was supported by a fellowship from the Irvington Institute for Medical Research. M. Cruz received support from the Hospital de Especialidades, Centro Medico Nacional Siglo XXI, Instituto Mexicano del Seguro Social (Mexico City, Mexico). R.A. Colbert received support as a Pfizer Scholar. The Basel Institute for Immunology was founded and is supported by F. Hoffmann-La Roche (Basel, Switzerland).
1.
|
Coux, O.,
K. Tanaka, and
A.L. Goldberg.
1996.
Structure and functions of the 20S and 26S proteasomes.
Annu. Rev.
Biochem.
65:
801-847
[Medline].
|
2.
|
Rock, K.L.,
C. Gramm,
L. Rothstein,
K. Clark,
R. Stein,
L. Dick,
D. Hwang, and
A.L. Goldberg.
1994.
Inhibitors of the
proteasome block the degradation of most cell proteins and
the generation of peptides presented on MHC class I molecules.
Cell.
78:
761-771
[Medline].
|
3.
|
Groll, M.,
L. Ditzel,
J. Löwe,
D. Stock,
M. Bochtler,
H.D. Bartunik, and
R. Huber.
1997.
Structure of 20S proteasome
from yeast at 2.4 Å resolution.
Nature.
386:
463-471
[Medline].
|
4.
|
Seemüller, E.,
A. Lupas,
D. Stock,
J. Löwe,
R. Huber, and
W. Baumeister.
1995.
Proteasome from Thermoplasma acidophilum: a threonine protease.
Science.
268:
579-582
[Medline].
|
5.
|
Martinez, C.K., and
J.J. Monaco.
1991.
Homology of proteasome subunits to a major histocompatibility complex-linked LMP gene.
Nature.
353:
664-667
[Medline].
|
6.
|
Glynne, R.,
S.H. Powis,
S. Beck,
A. Kelly,
L.-A. Kerr, and
J. Trowsdale.
1991.
A proteasome-related gene between the
two ABC transporter loci in the class II region of the human
MHC.
Nature.
353:
357-360
[Medline].
|
7.
|
Hisamatsu, H.,
N. Shimbara,
Y. Saito,
P. Kristensen,
K.B. Hendil,
T. Fujiwara,
E. Takahashi,
N. Tanahashi,
T. Tamura,
A. Ichihara, and
K. Tanaka.
1996.
Newly identified pair of
proteasomal subunits regulated reciprocally by interferon- .
J. Exp. Med.
183:
1807-1816
[Abstract].
|
8.
|
Nandi, D.,
H. Jiang, and
J.J. Monaco.
1996.
Identification of
MECL-1 (LMP-10) as the third IFN- -inducible proteasome subunit.
J. Immunol.
156:
2361-2364
[Abstract].
|
9.
|
Groettrup, M.,
R. Kraft,
S. Kostka,
S. Standera,
R. Stohwasser, and
P.-M. Kloetzel.
1996.
A third interferon- -induced subunit
exchange in the 20S proteasome.
Eur. J. Immunol.
26:
863-869
[Medline].
|
10.
|
Fruh, K.,
Y. Yang,
D. Arnold,
J. Chambers,
L. Wu,
J.B. Waters,
T. Spies, and
P.A. Peterson.
1992.
Alternative exon usage and
processing of the major histocompatibility complex-encoded proteasome subunits.
J. Biol. Chem.
267:
22131-22140
[Abstract/Free Full Text].
|
11.
|
Martinez, C.K., and
J.J. Monaco.
1993.
Post-translational processing of a major histocompatibility complex-encoded
proteasome subunit, LMP-2.
Mol. Immunol.
30:
1177-1183
[Medline].
|
12.
|
Akiyama, K.,
K. Yokota,
S. Kagawa,
N. Shimbara,
T. Tamura,
H. Akioka,
H.G. Nothwang,
C. Noda,
K. Tanaka, and
A. Ichihara.
1994.
cDNA cloning and interferon down-regulation of proteasomal subunits X and Y.
Science.
265:
1231-1234
[Medline].
|
13.
|
Gaczynska, M.,
K.L. Rock, and
A.L. Goldberg.
1993.
-interferon and expression of MHC genes regulate peptide hydrolysis by proteasomes.
Nature.
365:
264-267
[Medline].
|
14.
|
Boes, B.,
H. Hengel,
T. Ruppert,
G. Multhaup,
U.H. Koszinowski, and
P.-M. Kloetzel.
1994.
Interferon stimulation
modulates the proteolytic activity and cleavage site preference
of 20S mouse proteasomes.
J. Exp. Med.
179:
901-909
[Abstract].
|
15.
|
Ehring, B.,
T.H. Meyer,
C. Eckerskorn,
F. Lottspeich, and
R. Tampé.
1996.
Effects of major-histocompatibility-complex-encoded subunits on the peptidase and proteolytic activities of human 20S proteasomes.
Eur. J. Biochem.
235:
404-415
[Abstract].
|
16.
|
Gaczynska, M.,
K.L. Rock,
T. Spies, and
A.L. Goldberg.
1994.
Peptidase activities of proteasomes are differentially
regulated by the major histocompatibility complex-encoded
genes for LMP2 and LMP7.
Proc. Natl. Acad. Sci. USA.
91:
9213-9217
[Abstract/Free Full Text].
|
17.
|
Kuckelkorn, U.,
S. Frentzel,
R. Kraft,
S. Kostka,
M. Groettrup, and
P.-M. Kloetzel.
1995.
Incorporation of major histocompatibility complex-encoded subunits LMP2 and LMP7
changes the quality of the 20S proteasome polypeptide processing products independent of interferon- .
Eur. J. Immunol.
25:
2605-2611
[Medline].
|
18.
|
Gaczynska, M.,
A.L. Goldberg,
K. Tanaka,
K.B. Hendil, and
K.L. Rock.
1996.
Proteasome subunits X and Y alter peptidase activities in opposite ways to the interferon- -induced
subunits LMP2 and LMP7.
J. Biol. Chem.
271:
17275-17280
[Abstract/Free Full Text].
|
19.
|
Stohwasser, R.,
U. Kuckelkorn,
R. Kraft,
S. Kostka, and
P.-M. Kloetzel.
1996.
20S proteasome from LMP7 knock out mice
reveals altered proteolytic activities and cleavage site preferences.
FEBS Lett.
383:
109-113
[Medline].
|
20.
|
Van Kaer, L.,
P.G. Ashton-Rickardt,
M. Eichelberger,
M. Gaczynska,
N. Nagashima,
K.L. Rock,
A.L. Goldberg,
P.C. Doherty, and
S. Tonegawa.
1994.
Altered peptidase and viral-specific T cell response in Lmp2 mutant mice.
Immunity.
1:
533-541
[Medline].
|
21.
|
Fehling, H.J.,
W. Swat,
C. Laplace,
R. Kühn,
K. Rajewsky,
U. Müller, and
H. von Boehmer.
1994.
MHC class I expression in mice lacking the proteasome subunit LMP-7.
Science.
265:
1234-1237
[Medline].
|
22.
|
Nandi, D.,
E. Woodward,
D.B. Ginsburg, and
J.J. Monaco.
1997.
Intermediates in the formation of mouse 20S proteasomes: implications for the assembly of precursor subunits.
EMBO (Eur. Mol. Biol. Organ.) J.
16:
5363-5375
[Abstract/Free Full Text].
|
23.
|
Frentzel, S.,
B. Pesold-Hurt,
A. Seelig, and
P.-M. Kloetzel.
1994.
20 S proteasomes are assembled via distinct precursor
complexes.
J. Mol. Biol.
236:
975-981
[Medline].
|
24.
|
Yang, Y.,
K. Früh,
K. Ahn, and
P.A. Peterson.
1995.
In vivo
assembly of the proteasomal complexes, implications for antigen processing.
J. Biol. Chem.
270:
27687-27694
[Abstract/Free Full Text].
|
25.
|
Patel, S.D.,
J.J. Monaco, and
H.O. McDevitt.
1994.
Delineation of the subunit composition of human proteasomes using
antisera against the major histocompatibility complex-encoded
LMP2 and LMP7 subunits.
Proc. Natl. Acad. Sci. USA.
91:
296-300
[Abstract].
|
26.
|
Hendil, K.B.,
P. Kristensen, and
W. Uerkvitz.
1995.
Human proteasomes analysed with monoclonal antibodies.
Biochem. J.
305:
245-252
[Medline].
|
27.
|
Salter, R.D.,
D.N. Howell, and
P. Cresswell.
1985.
Genes regulating HLA class I antigen expression in T-B lymphoblastoid hybrids.
Immunogenetics.
21:
235-246
[Medline].
|
28.
|
Ortiz-Navarette, V.,
A. Seelig,
M. Gernold,
S. Frentzel,
P.-M. Kloetzel, and
G.J. Hämmerling.
1991.
Subunit of the `20S'
proteasome (multicatalytic proteinase) encoded by the major
histocompatibility complex.
Nature.
353:
662-664
[Medline].
|
29.
|
Groettrup, M.,
S. Standera,
R. Stohwasser, and
P.-M. Kloetzel.
1997.
The subunits MECL-1 and LMP2 are mutually required for incorporation into the 20S proteasome.
Proc. Natl.
Acad. Sci. USA.
94:
8970-8975
[Abstract/Free Full Text].
|
30.
|
Seemüller, E.,
A. Lupas, and
W. Baumeister.
1996.
Autocatalytic processing of the 20S proteasome.
Nature.
382:
468-470
[Medline].
|
31.
|
Chen, P., and
M. Hochstrasser.
1996.
Autocatalytic subunit
processing couples active site formation in the 20S proteasome to completion of assembly.
Cell.
86:
961-972
[Medline].
|
32.
|
Schmidtke, G.,
R. Kraft,
S. Kostka,
P. Henklein,
C. Frömmel,
J. Löwe,
R. Huber,
P.-M. Kloetzel, and
M. Schmidt.
1996.
Analysis of mammalian 20S proteasome biogenesis: the
maturation of -subunits is an ordered two-step mechanism
involving autocatalysis.
EMBO (Eur. Mol. Biol. Organ.) J.
15:
6887-6898
[Abstract].
|
33.
|
Stohwasser, R.,
S. Standera,
I. Peters,
P.-M. Kloetzel, and
M. Groettrup.
1997.
Molecular cloning of the mouse proteasome subunits MC14 and MECL-1: reciprocally regulated
tissue expression of interferon- -modulated proteasome subunits.
Eur. J. Immunol.
27:
1182-1187
[Medline].
|
34.
|
Nandi, D.,
M.N. Iyer, and
J.J. Monaco.
1996.
Molecular and
serological analysis of polymorphisms in the murine major histocompatibility complex-encoded proteasome subunits,
LMP-2 and LMP-7.
Exp. Clin. Immunogenet.
13:
20-29
[Medline].
|