(Received for publication, June 30, 1995)
From the
The multicatalytic and multisubunit proteasomal complexes have
been implicated in the processing of antigens to peptides presented by
class I major histocompatibility complex molecules. Two structural
complexes of this proteinase, 20 S and 26 S proteasomes, have been
isolated from cells. By analyzing in vivo assembly of the
proteasomal complexes we show that the 20 S proteasomal complexes are
irreversibly assembled via 15 S assembly intermediates containing
unprocessed -type subunits. The 20 S proteasomes further associate
reversibly with proteasome activators PA28 or pre-existing ATPase
complexes to form 26 S proteasomal complexes. Our findings that not all
of the 20 S proteasomal complexes are assembled into 26 S proteasomal
complexes within cells and that all of PA28 and ATPase complexes are
associated with 20 S proteasomes strongly suggest that all proteasomal
complexes coexist within cells. We further demonstrate that 26 S
proteasomal complexes are predominantly present in the cytoplasm and a
significant portion of the 20 S proteasomal complexes is associated
with the endoplasmic reticulum membrane. Taken together, our findings
suggest that depending upon their associated regulatory components, 26
S and 20 S-PA28 proteasomal complexes serve different housekeeping
functions within the cells, while they degrade antigens in a
cooperative manner in antigen processing.
Cytotoxic T-lymphocytes recognize antigenic peptides of
8-10 amino acid residues presented by class I major
histocompatibility complex (MHC) ()molecules(1) .
This trimeric complex of class I MHC molecule is assembled in the
endoplasmic reticulum from class I heavy chain,
-microglobulin, and antigenic peptide imported from
the cytoplasm through a peptide
transporter(2, 3, 4) . Because antigen
exogenously introduced into the cytoplasm needs to be ubiquitinated
before T-cell epitopes are generated(5) , it seems that
antigenic peptide generation follows a ubiquitin-mediated protein
degradation pathway. It has been shown that ubiquitinated proteins are
degraded by the 26 S multicatalytic and multisubunit proteinase
complex, or 26 S proteasome (6) and that inhibitors of the
proteasomal complexes block the generation of peptides presented on
class I MHC molecules(7) .
The 26 S proteasome consists of
an ``ATPase complex'' (8) (or ``19 S
complex''(9) ) and the 20 S proteasome which is believed
to be the catalytic core of the 26 S
proteasome(10, 11, 12) . The 20 S proteasome
is a highly conserved structure of 7-fold symmetry in all eucaryotic
cells and the archaebacterium Thermoplasma
acidophilum(10, 11, 12) . The x-ray
crystallographic analysis of the 20 S proteasome from the
archaebacterium T. acidophilum has recently been
reported(13) . The 20 S proteasome of eucaryotes consists of a
family of 14 different subunits of molecular masses between 20 and 35
kDa(14, 15) . The subunits can be classified into
seven different but homologous -type or
-type subunits
according to their homology with the
- and
-type subunits of
the T. acidophilum proteasome(16, 17) . In addition, three
additional
-type subunits, LMP2, LMP7, and MECL1, are up-regulated
by interferon
(IFN
)(15, 18, 19, 20, 21, 22, 23, 24) .
The MHC-encoded and IFN
-inducible LMP2 and LMP7 (23) are
found to displace two constitutively expressed and highly homologous
housekeeping
-type subunits 2 and 10, respectively, from the
proteasomal complexes(23, 24) , thus changing the
subunit composition without altering the number of subunits per
complex. Although incorporation of LMP2 and LMP7 is not an essential
prerequisite for peptide generation(25, 26) , the
enzymatic activities of proteasomal complexes are thereby altered in
such a way that preferred peptidic substrates for peptide transporters
and class I molecules are generated (27, 28, 29, 30, 31) . (
)Further circumstantial evidence for the involvement of
proteasomal complexes in antigen processing came from studies of a
family of homologous proteasome activators PA28 (32, 33, 34, 35) which consists of
at least two IFN
-inducible homologues.
It has been
shown that in vitro binding of PA28 to 20 S proteasomes
activates distinct peptidase activities of 20 S proteasomes (32, 33, 34) ,
indicating that
peptide products appropriate for antigen presentation may be
preferentially generated.
Both 20 S and 26 S proteasomes can be
isolated from
cells(11, 12, 14, 18) , but it has
not been clearly established whether both complexes coexist in
vivo. We have therefore studied the individual steps leading to
the formation of 26 S proteasomal complexes. We show that -type
subunit precursors assemble into distinct assembly intermediates prior
to processing, whereby the amino-terminal sequences play a crucial
role. The assembly of 20 S proteasomes is completed with the
association of additional subunits to the assembly intermediate during
and/or after
-type subunits are processed, whereas the assembly of
26 S proteasomal complexes occurs in a single step from the
pre-existing ATPase complexes and 20 S proteasomes. Furthermore, we
present the first in vivo observation that IFN
-inducible
proteasome activators PA28 directly bind to 20 S proteasomes and that
binding of PA28 does not interfere with the assembly of 26 S
proteasomal complexes. Although the assembly of 20 S proteasomes is
irreversible, assembly of the 26 S proteasomal complexes, possibly
mediated by subunit phosphorylation, is reversible. Our demonstration
that 20 S and 26 S proteasomal complexes localize in different
subcellular compartments further suggests the differential functions of
20 S and 26 S proteasomal complexes in antigen processing.
Figure 2: Assembly of 20 S proteasomal complexes. Proteasomes were immunoprecipitated from sucrose gradient fractions of homogenates prepared from RMA cells after labeling for 30 min (A) and chasing for 2 h (B). The antiserum used was raised against recombinant subunit C9 and precipitates both free (fractions 1-2) and complexed (fractions 3-12) subunit C9(22) . The positions of the MHC-encoded subunits LMP2 and LMP7 as well as their precursors are marked. Sedimentation coefficients for fractions 5 and 9 were approximately 15 S and 20 S, respectively.
Figure 4:
Assembly of 26 S proteasomal complexes.
RMA cells were labeled for 30 min (A) followed by 2 h chase (B), 6 h chase (C), or 12 h chase (D).
Fractionation of RMA cell homogenates was performed as in the legend to Fig. 2, except that 2 mM ATP and 5 mM
MgCl were included in cell homogenization isotonic solution
and sucrose gradients. Subunits smaller than 32 kDa belong to the 20 S
proteasomes (see Fig. 2), subunits larger than this molecular
mass are part of the ATPase complexes (see Fig. 5). The
sedimentation coefficient for the complexes at fraction 11 was
approximately 26 S. Some of the proteasomal subunits as well as their
precursors are indicated. The
C-methylated protein markers (M) are myosin (200 kDa), phosphorylase b (97.4 kDa),
bovine serum albumin (69 kDa), ovalbumin (46 kDa), carbonic anhydrase
(30 kDa), and lysozyme (14.3 kDa). E, the enzymatic activities
of the different proteasomal complexes present in each sucrose gradient
fractions were assayed for hydrolysis of the fluorogenic peptidic
substrate succinyl-LLVY-AMC.
Figure 5: ATPase complexes associate rapidly with pre-existing 20 S proteasomes. RMA cell homogenates were fractionated as described in the legend to Fig. 4except that RMA cells were labeled for 1 h (A and C) followed by a 4-h chase (B and D) prior to fractionation and immunoprecipitation with anti-ATPase complex antiserum (A and B) or anti-C9 antiserum (C and D). Only fractions 5-14 are shown. Sedimentation coefficients for fractions 5, 8, and 11 were approximately 15 S, 20 S, and 26 S, respectively. Note that the ATPase complexes associate with unlabeled 20 S proteasomes under these labeling conditions (compare A and C). A similar pattern to C was also observed for Fig. 4A upon longer exposure of the fluorogram. Proteins immunoprecipitated by the anti-ATPase complex antiserum in fractions other than 9-12 are most likely nonspecific, since they remained unchanged during the chase period (compare A and B).
Figure 6:
Proteasome activators PA28 bind to 20 S
proteasomes. The expression of IFN-inducible PA28 in the stable
PA28-expressing HtTa transfectant cells was induced by the removal of
tetracycline for 16 h prior to [
S]methionine
labeling for 30 min followed by a 4-h chase. Fractionation of cell
homogenates was performed as described in the legend to Fig. 4.
Each fraction was equally divided into two aliquots and
immunoprecipitated either with anti-proteasome (left panel) or
anti-PA28 antisera (right panel).
Figure 1:
Incorporation of -type subunit
LMP7 into proteasomal complexes. HtTA cells were either transfected
with murine LMP7 (lanes 1 and 2), human LMP7-E2 (lanes 3 and 4), human LMP7-E1 (lanes 5 and 6), or un-transfected but induced (lanes 8 and 9) or uninduced (lane 7) with IFN
. After
[
S]methionine metabolic labeling, cells were
lysed and proteins were immunoprecipitated with either anti-LMP7
antiserum (22) (lanes 1, 3, 5, and 8) or
anti-proteasome antiserum (24) (lanes 2, 4, 6, 7, and 9). The positions of precursors and processed products of
human and mouse LMP7 are indicated.
When
we immunoprecipitated the proteasomal complexes, protein bands
corresponding to the processed forms of LMP7 were present only in
proteasomal complexes from cells transfected with either human LMP7-E2 (Fig. 1, lane 4) or mouse LMP7 (lane 2) but
not human LMP7-E1 (lane 6). No processed LMP7-E1, which should
be identical to processed transfected- or IFN-induced endogenous
LMP7-E2, was detected in overexposed fluorograms either (not shown);
thus, it seems that only LMP7-E2 gives rise to a functional protein. We
conclude that the amino-terminal prosequence is crucially involved in
the mechanism by which
-type subunits are assembled into the
proteasomes. Our finding in conjunction with the observations that the
prosequences differ significantly among different
-type subunits
and that the sequences surrounding the cleavage site (17, 24) are highly conserved suggest that the
prosequences might play a subunit-specific role in the initial folding
and/or assembly of
-type subunits (see below), whereas the
cleavage is performed by a specific protease.
In order to determine whether there are differences among
these complexes, we compared the subunit composition of the
``anti-LMP2 15 S complexes'' with the ``anti-C9 15 S
complexes'' as well as with the 20 S complexes by two-dimensional
gel electrophoresis. As shown in Fig. 3, the subunit
compositions of 15 S complexes immunoprecipitated by anti-C9 or LMP2
antisera were almost identical but differed in a number of subunits
from the 20 S complex and the 20 S complexes displayed the typical
two-dimensional pattern of 20 S proteasomes(23) . Furthermore,
subunits common to all three complexes were identified as -type
subunits by using a panel of subunit-specific antisera and
two-dimensional immunoblots as described (22) . Interestingly,
-type subunit C3 displayed a charge shift in the anti-LMP2 15 S
complexes indicating post-translational modifications. Among the
subunits differing between 15 S and 20 S complexes, four subunits in
the 15 S complexes could be identified as precursors of the
-type
subunits, pC5, pLMP2, pLMP7, and an unidentified
-type subunit p,
whereas their processed forms are only present in the 20 S proteasomes (Fig. 3B). Since
-type subunit precursors are only
present in the 15 S assembly intermediates, we conclude that
-type
subunits assemble into 15 S complexes prior to their processing.
Figure 3:
Subunit compositions of 15 S assembly
intermediates and 20 S proteasomal complexes. The same sucrose gradient
fractions 5 (C and D) and 9 (B) from Fig. 2were immunoprecipitated with either anti-C9 (B and D) or anti-LMP2 (C) antisera (22) and immunoprecipitates were resolved according to charge
and molecular mass(23) . A, schematic summary of the
subunit composition common to (black) or different between (open and dotted) the 20 S proteasomal complexes (B) and the 15 S assembly intermediates (D). Compared
to the subunit composition of the anti-C9 15S complexes (D),
two subunits indicated with arrows are absent from the
anti-LMP2 15 S complexes (C). Subunits or their precursors
(``p'') identified in two-dimensional immunoblots with a
panel of subunit-specific antisera (22) are indicated. It is
conceivable that the seven subunits (black) common to the 20 S
and 15 S complexes are -type subunits and that
-type subunit
precursors are allowed to associate with the 7-fold structural backbone
composed of
-type subunit, thus forming 15 S
complexes.
The
almost identical subunit composition and half-life of both the 15 S
assembly intermediates recognized by anti-C9 and anti-LMP2 antisera as
well as similar complexes observed by Frentzel et al.(37) and Patel et al.(38) indicates a
time-limited step at this point of proteasomal complex assembly. Our
data further show that 20 S proteasome assembly is an irreversible
process, because free processed -type subunits could not be
detected within the cells(24) . In addition, our findings that
no
-type subunit precursors were detected in 20 S complexes and no
intermediate assembly complexes of
rings (see below) were
detected suggest that the completion of 20 S proteasomal complex
assembly, i.e. prosequence cleavage and dimerization of the
two
rings, occurs rather rapidly. We conclude that due to
this irreversible process, homologous subunit exchanges can only take
place during assembly of the 15 S complexes, as we have indeed observed
for LMP subunits(24) . Interestingly, although both LMP2 and
subunit 2 are present in 20 S proteasomes of RMA cells, indicating a
mixed proteasome population, only the LMP2 precursor is detected in the
15 S assembly intermediates immunoprecipitated by either anti-C9 or
anti-LMP2 antisera. This suggests that the order of assembly with other
subunits is different for LMP2 than for subunit 2.
Because the anti-ATPase complex polyclonal antiserum
is raised against isolated ATPase complexes, it is unlikely that this
antiserum does not recognize free ATPase complexes. Our data indicating
that no preformed free ATPase complexes were detected in other
fractions (not shown) also suggests that the amount of the ATPase
complexes is limiting in cells and that these complexes associate
rapidly with the pre-existing 20 S proteasomal complexes. These
findings in conjunction with the observation that only about 40% of the
label present in the 20 S complexes is converted to the 26 S complexes
even after prolonged chase (Fig. 4D) further suggests
that the free 20 S proteasomal complexes coexist with 26 S proteasomal
complexes within the cells and are not the dissociation products of 26
S complexes. We next studied the enzymatic activities of the different
proteasomal complexes present in sucrose gradient fractions for
hydrolysis of the succinyl-LLVY-AMC, a fluorogenic peptidic substrate
commonly used for detection of proteasome
activity(11, 12) . The enzymatic activities were only
detected in fractions containing the 20 S and 26 S complexes, whereas
no enzymatic activity was observed for the fractions containing free
subunits or 15 S assembly intermediates (Fig. 4E). This
result indicates that only after cleavage of the -type subunit
prosequences are proteasomal complexes enzymatically active.
Figure 7: Distinct subcellular distribution of 20 S and 26 S proteasomal complexes. Cell homogenates prepared from RMA cells labeled for 0.5 h (odd-numbered lanes) followed by a 2-h chase (even-numbered lanes) were fractionated into three subcellular fractions by differential centrifugation: nuclei (lanes 1 and 2), cytosol (lanes 3 and 4), and microsomes (lanes 5 and 6). Immunoprecipitations were performed by using anti-C9 (left panel) or anti-LMP2 (right panel) antisera.
Figure 8:
Regulation of proteasomal complex
assembly. A, some subunits of 20 S proteasomes and ATPase
complex are phosphorylated. RMA cells were lysed in 1% Nonidet P-40
lysis buffer containing phosphatase inhibitors (1 µM okadaic acid, 0.1 µM calyculin A, 50 mM NaF,
30 mM pyrophosphate, and 0.1 mM
NaVO
) after labeling with either
[
P]orthophosphate (lanes 1 and 2) or [
S]methionine (lane 3).
26 S complexes from cell lysates were immunoprecipitated using the
anti-C9 antiserum. Predominantly phosphorylated subunits are indicated.
In lane 2 cells had been incubated with 1 µM okadaic acid and 0.1 µM calyculin A during labeling. B, effect of okadaic acid/calyculin A on proteasomal complex
assembly. RMA cells were untreated (lanes 1 and 2) or
treated (lanes 3 and 4) with 1 µM
okadaic acid and 0.1 µM calyculin A for 0.5 h prior to and
during labeling (0.5 h). Proteasomes were either directly (lanes 1 and 3) or after 4-h chase (lanes 2 and 4) immunoprecipitated with anti-LMP2 antiserum. C,
effect of cycloheximide on proteasomal complex assembly. RMA were
induced (lanes 2 and 4) or uninduced (lanes 1 and 3) by IFN
for 48 h prior to 30 min metabolic
labeling. After labeling, cells were cultured for an additional 1 h in
the absence (lanes 1 and 2) or presence (lanes 3 and 4) of 1 µM cycloheximide. Proteasomes
were immunoprecipitated directly using the anti-C9 antiserum. D, staurosporine inhibits assembly of 26 S complexes. RMA
cells were labeled for 4 h and the label was chased for 0 (lanes 1 and 7), 24 (lanes 2 and 8), 48 (lanes 3 and 9), 72 (lanes 4 and 10), 96 (lanes 5 and 11), and 120 h (lanes 6 and 12) either in the absence (left
panel) or presence (right panel) of 0.5 µM staurosporine.
Since no phosphorylated subunits were detected in 15 S assembly
intermediates by in vivoP labeling followed by
immunoprecipitation with anti-LMP2 antiserum (data not shown), it is
unlikely that phosphorylation plays a role in assembly of the 15 S
assembly intermediates. We therefore studied whether phosphorylation
and dephosphorylation would have an effect on the assembly of the 20 S
and 26 S proteasomal complexes. RMA cells were either treated in
vivo with the serine/threonine phosphatase inhibitors okadaic acid
and calyculin A (Fig. 8B) or with the kinase inhibitor
staurosporine (Fig. 8D). In addition, we analyzed the
effect of cycloheximide (Fig. 8C) on the proteasomal
complex assembly. As shown in Fig. 8C, the
cycloheximide treatment resulted in drastically reduced assembly of the
20 S proteasome. Since under this condition the subunit composition of
anti-C9 immunoprecipitates (only subunit 7 (23) coprecipitated
significantly with the subunit C9) is simpler than 15 S assembly
intermediates, we conclude that cycloheximide blocked assembly at a
very early stage prior to 15 S complex formation, presumably due to the
lack of newly synthesized subunits. By contrast, treatment by okadaic
acid and calyculin A did not inhibit the assembly of the 15 S complexes
but resulted in decreased processing of these assembly intermediates as
shown by immunoprecipitating the 15 S complexes with anti-LMP2
antiserum (Fig. 8B). Similarly, a decreased rate of 20
S complex formation was also observed, when anti-C9 antiserum was used
for immunoprecipitation (data not shown). Furthermore, when the kinase
inhibitor staurosporine was used, the anti-C9 antiserum no longer
coprecipitated the ATPase complexes with the 20 S complexes (Fig. 8D, right panel). We ruled out that this effect
was due to an increased turnover of 26 S proteasomes, since after a
prolonged chase period of several days no noticeable difference in the
half-life of 20 S proteasomes between staurosporine-treated and
untreated cells was observed (Fig. 8D). Consistent with
the observation that in vitro the 26 S proteasome can be
reversibly disassembled into the ATPase complex and 20 S complex by
removal of ATP/Mg(11, 12) , we found that upon removal
of staurosporine 26 S proteasomes were again detectable.
This effect of staurosporine indicates that kinases play a role
in the assembly and disassembly of the 26 S complexes. Since
staurosporine treatment gives a similar result in vivo, we
propose that the role of ATP in 26 S complex assembly is dependent on
an ATP-dependent protein kinase. It is interesting to note that PA28 is
phosphorylated(42) , suggesting that phosphorylation may
account for the association with the 20 S proteasomal complexes both
for the ATPase complex and PA28.
Based on studies of the assembly and structure of Thermoplasma proteasomes (44, 13) and our
findings presented in this paper, a hypothetical model for the in
vivo assembly of 26 S complexes is proposed in Fig. 9.
Eucaryotic -type subunits assemble on a ring-like structural
backbone composed of seven
-type subunits. Thus, seven
-type
subunits first associate among each other to form a ring-like
structural backbone of seven-fold symmetry in which seven
-type
subunits are allowed to assemble. It is conceivable that each
-type subunit occupies a particular position within the ring-like
structure and each
-type subunit precursor also assembles in a
given order by binding to the corresponding
-type subunits and
each other. This process may lead to proteasomal complexes differing in
the content of their exchangeable subunit homologues, e.g. LMP2 and LMP7. The
-type subunit prosequences are immediately
cleaved off either before or during the dimerization of the two
-ring structural complexes. During assembly of
proteasomal intermediate complexes, the prosequences of
-type
subunits might play a role in either the protein folding itself or the
proteasome assembly process or by inactivating the enzymatic activity
of the catalytic sites created during assembly. Thus, seven
-type
and seven
-type subunits each form a ring-like structure of 7-fold
symmetry which further assembles into a dimer consisting of four-ring
cylindrical structure in the order
, i.e. the 20 S proteasomal
complexes(11, 12, 13, 18) . The 20 S
proteasomal complexes are then bound to regulatory components such as
the ATPase complexes (16, 18) or the
PA28(32, 33, 34, 35) . The 20 S
proteasomal complexes are capped end-on by the ATPase complexes to form
the 26 S complexes (16) or by the PA28(39) .
Association with these regulatory components appears to be reversible.
Once bound to the regulatory components the enzymatic activities of
proteasomal complexes are activated.
Figure 9:
Proposed assembly pathway of the
proteasomal complexes. Seven -type subunits first associate among
each other to form a ring-like structural backbone of 7-fold symmetry
in which seven
-type subunits are allowed to assemble. Thus, seven
-type and seven
-type subunits each form a ring-like
structure of 7-fold symmetry which further assembles into a dimer
consisting of a four-ring cylindrical structure in the order
, i.e. the 20 S complexes. The 20 S
proteasomal complexes are then capped end-on to regulatory components
such as the PA28 or the ATPase complexes, thus forming the 26 S
complexes. Association with these regulatory complexes appears to be
reversible and probably regulated by
phosphorylation.
The observations that upon
induction of PA28 by IFN the molar ratios of PA28 and 20 S
proteasomes, as well as ATPase complexes and 20 S proteasomes, are
approximately 1:50 and 2:5, respectively,
supports the
notion that there is a large pool of 20 S proteasomes within cells and
that proteasome activities are modulated by regulatory components. It
is conceivable that all 20 S proteasome-derived complexes serve
specific purposes. The 26 S complexes seem to perform housekeeping
functions, i.e. ubiquitin-dependent protein degradation, since
the ATPase complexes are shown to be responsible for ubiquitin
recognition and cleavage as well as regulatory functions(12) .
The 20 S or 20 S-PA28 proteasomal complexes appear to cleave
low-molecular mass peptidic substrates such as degradation
intermediates, since 20 S or 20 S-PA28 complexes are rather inefficient
in degrading whole proteins, even ubiquitinated
proteins(12, 18, 32, 33, 34) .
Since we found that all proteasome subunits, including the MHC-encoded
LMP subunits, found in the 20 S proteasomal complexes were also found
in the 26 S proteasomal complexes (Fig. 4) and that the ratios
of LMP subunits to their exchanging homologous subunits remained the
same between 20 S and 26 S complexes, we conclude that expressions of
MHC-encoded LMP subunits affect both 20 S and 26 S proteasomal
complexes. Therefore, our observations that subsets of 20 S complexes,
differing with regard to their subunit composition, are also reflected
in the subsets of 26 S complexes and that both 20 S-PA28 and 26 S
complexes are enzymatically active suggest that both 20 S-PA28 and 26 S
complexes may be involved in antigen processing. Our observations that
peptide loading onto class I MHC molecules is interfered with by
okadaic acid/calyculin A treatment(46) ,
which
inhibits the in vivo assembly of 20 S proteasomal complexes,
and that upon expression of PA28 the intracellular transport rate of
class I MHC molecules increases 25%
in conjunction with our
finding that IFN
-inducible PA28 directly binds to 20 S proteasomes
without interfering with the assembly of 26 S proteasomal complexes
strongly suggest that 20 S-PA28 proteasomal complexes play a direct
role in antigen processing. Moreover, our finding that at least two
PA28 homologues are up-regulated by IFN
further
supports the above hypothesis. It is interesting to note that isolated
20 S proteasomes of T. acidophilum have been shown to
cleave small protein substrates into peptides with a length
distribution centering around 8 amino acids(45) . Thus, it is
reasonable to suggest that IFN
-inducible LMP and PA28 serve to
optimize antigen processing in proteasome-mediated protein degradation.
With regard to antigen processing for MHC class I antigen
presentation, several parallel pathways could co-exist. 26 S
proteasomal complexes could be responsible for antigenic peptide
generation from ubiquitinated antigens, whereas 20 S or 20 S-PA28
proteasomal complexes could digest either partially unfolded antigens
or newly synthesized abnormal polypeptides to antigenic peptides. This
notion is consistent with the findings that 26 S proteasomal complexes
but not 20 S or 20 S-PA28 proteasomal complexes are responsible for the
degradation of ubiquitinated proteins and that distinct peptidase
activities of 20 S proteasomes can be activated by IFN-inducible
PA28. Alternatively, antigen degradation could occur in several
successive steps conducted by various proteasomal complexes. 26 S
proteasomal complexes could be the protease to degrade whole antigen
into degradation intermediates and 20 S-PA28 complexes could function
in further processing of such degradation intermediates into peptides
of 8-10 amino acid residues suitable for class I loading. Based
on our findings that 26 S proteasomal complexes are predominantly
present in the cytoplasm and a significant portion of the 20 S
proteasomal complexes is associated with endoplasmic reticulum
membrane, it can be envisaged that after ubiquitinated proteins are
fragmented by the 26 S proteasomal complexes, final trimming to the
size of peptides required for transport and class I binding is perfomed
by the 20 S-PA28 complexes in close spatial vicinity to the peptide
transporters which are localized in the endoplasmic reticulum membrane.
The direct association of 20 S proteasomal complexes and
transporter-associated with antigen processing in the endoplasmic
reticulum membrane could be an efficient means of transporting peptides
generated by 20 S proteasomal complexes to transporter-associated with
antigen processing. Our finding that no MHC class I peptide loading
occurs
if the ubiquitination pathway is blocked (5) strongly supports the latter hypothesis.