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J Biol Chem, Vol. 275, Issue 9, 6592-6599, March 3, 2000
,From the Instituto de Investigaciones Biomédicas Alberto Sols, CSIC-UAM, Facultad de Medicina de la Universidad Autónoma de Madrid, 28029 Madrid, Spain
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Assembly of mammalian 20 S proteasomes from
individual subunits is beginning to be investigated. Proteasomes are
made of four heptameric rings in the configuration The 20 S proteasome is the enzyme responsible for most
non-lysosomal protein degradation in eukaryotes, and structural
homologues are present in archeons and eubacteria (1, 2). The overall structure of the proteasome is a hollow cylinder composed of four heptameric rings in the configuration Most of the The assembly of the Thermoplasma proteasome has been
elucidated (6). The precursor Research into the pathway of mammalian proteasome assembly is just
beginning. Intermediates with sedimentation coefficients of 13 S,
15 S, and 16 S have been described (12, 14, 15, 17). Complete
pre-proteasomes (13 S, 15 S) have been postulated to be
"half-proteasomes" composed of a full The C5 component of the proteasome belongs to the Cell Culture and Antisera--
NRK (rat), CHO (hamster), and
HeLa (human) cells were cultured in Dulbecco's modified Eagle's
medium supplemented with 10% fetal calf serum and grown to 60-80%
confluence. The rabbit anti-proteasome and anti-C5-specific antibodies,
as well as other anti-subunit-specific antibodies (anti-C2, -C8, and
-C9), have been described (22, 25, 26).
Preparation of Subcellular Fractions--
NRK cells (70%
confluent) were cooled on ice and washed 3 times with cold PBS. All
subsequent steps were performed at 4 °C. Cells were scraped in PBS,
centrifuged, and the pellet lysed by up and down pipetting in a buffer
containing 10 mM Tris-Cl, pH 8.0, 7.5 mM
(NH4)2SO4, 1 mM EDTA,
0.025% Nonidet P-40, and 1 mM dithiothreitol. After
incubation on ice for 5 min, sucrose was added to the homogenate (0.3 M final concentration). Complete cell lysis was checked by
phase contrast microscopy and trypan blue staining. Subcellular
fractions were obtained by differential centrifugation of the cell
homogenate as follows: nuclei, pellet of the cell homogenate after
centrifugation at 10,000 × g for 20 s;
mitochondria, pellet of the nuclear supernatant after centrifugation at
10,000 × g for 10 min; microsomes and cytosol (S100),
pellet and supernatant of the post-mitochondrial supernatant after
centrifugation at 100,000 × g for 60 min,
respectively. All pellet fractions were washed once with lysis buffer
containing sucrose by gentle resuspension and recentrifugation as
indicated above. Approximately 50 µg of protein of each of the
subcellular fractions was used for immunoblot analysis with the
indicated antibodies.
Characterization of Anti-C5-, Anti-C8-, and Anti-C9-specific
Antibodies and Protein Analysis--
Rat liver proteasome was purified
as described (27, 28). The anti-subunit-specific antibodies were
characterized by immunoprecipitation of the purified rat liver
proteasomes under native and denaturing conditions. Native conditions
for purified proteasomes in immunoprecipitation buffer are as follows:
TBS (50 mM Tris-Cl, pH 7.5, 150 mM NaCl) containing 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl
fluoride, and 10 µg/ml of leupeptin. Denaturing conditions for
purified proteasome were in the same buffer as above but containing
0.1% SDS and were boiled for 2 min before immunoprecipitation. For ELISA, 50 µl of a solution containing 50 µg/ml purified rat liver proteasome in TBS was used to coat 96-well plates (Nunc) by incubation overnight at 4 °C. Plates were then washed with TBS and blocked for
3 h at room temperature with 200 µl of blocking buffer (TBS with
3% bovine serum albumin) containing 0.1% Tween 20. Sera (50 µl) at
different dilutions in blocking buffer with detergent were added to the
wells and incubated for 3 h at room temperature or overnight at
4 °C. After washing with TBS, the secondary peroxidase-labeled antibody at 1/1000 dilution in blocking buffer with detergent was added
and incubated for 1 h at room temperature. After extensive washes,
reaction was developed and quantitated in an EL340 (from Biotek)
microplate reader at 490 nm. In each experiment, assays were run in
triplicate, and control wells containing no primary antibody or
preimmune sera were included to subtract background.
Immunoprecipitations were performed with the indicated antibodies
previously coupled to protein A-Sepharose (Sigma) by incubation in TBS
for 2 h at room temperature with rocking and washed 5 times with
immunoprecipitation buffer (see above) by spinning in an Eppendorf
microcentrifuge (10,000 rpm for 15 s). The samples to be
immunoprecipitated were added to the beads containing the coupled antibodies and incubated at 4 °C for 3 h with rocking. The
beads were washed three times with 1 ml of immunoprecipitation buffer (by spinning in an Eppendorf centrifuge, as above) and once with distilled water. The proteins were eluted with SDS-sample buffer and
analyzed by SDS-PAGE.1
Proteins were analyzed on 10-20% gradient or 13% continuous
SDS-PAGE. After electrophoresis, the gels were either stained with
Coomassie Blue or transferred to nitrocellulose filters and processed
for immunoblot analysis as described (22). The anti-subunit-specific antibodies were used at 1/200-1/500 dilution, and the immunoblots were
developed with an alkaline phosphatase-labeled goat anti-rabbit antibody (Bio-Rad) at 1/1000 dilution.
In Vitro Transcription of C5 cDNA and Translation in Rabbit
Reticulocyte Lysates--
The pTrC5 (rat) or pTmC5 (mouse) plasmids
(22) were linearized by digestion with HindIII (3' cleavage)
and purified from low melting point agarose gels followed by Geneclean,
as described by the manufacturer (Bio 101). The in vitro
transcription reaction contains the following in a final volume of 50 µl: 50 mM Tris-Cl, pH 7.5, 1 mM spermidine, 5 mM MgCl2, 1 mM dithiothreitol, 2 units of ribonuclease A inhibitor (RNasin, Amersham Pharmacia Biotech), 1 mM 7-methyl-GpppG (Roche Molecular Biochemicals), 1 mM each of ATP, CTP, and UTP; 0.2 mM GTP, 2 µg of linearized pTrC5 or pTmC5, and 10 units of T7-RNA polymerase
(Roche Molecular Biochemicals). The transcription reaction was
incubated at 37 °C for 2 h, stopped by addition of EDTA to 10 mM (final concentration), extracted once each with phenol,
phenol:chloroform (1:1), and chloroform, and precipitated with ethanol.
The transcribed mRNA was translated in a rabbit reticulocyte lysate
in the presence of 20 µCi of [35S]methionine/cysteine
(Translabel, ICN) for 1 h at 30 °C according to the protocol of
the lysate manufacturer (Amersham Pharmacia Biotech). The translation
products were analyzed by 13% SDS-PAGE after stopping the reaction
with SDS-sample buffer and boiling for 3 min. For immunoprecipitation,
the translation reactions were stopped by dilution with 1 ml of cold
immunoprecipitation buffer and centrifuged at 15,000 × g for 20 min at 4 °C. The supernatants were used for
immunoprecipitation, as described above.
Metabolic Labeling of Cells--
Cells (NRK, HeLa, and CHO) were
metabolically labeled with 250 µCi/ml
[35S]methionine/cysteine (Translabel, ICN) for 30 min (or
3 h, pulse) in Dulbecco's modified Eagle's medium without
methionine/cysteine and then chased for different times with complete
medium. In some experiments cells were treated with 5 µM
lactacystin (Calbiochem) during the pulse and chase periods. At the
times indicated, cells were washed with cold PBS (3 times), lysed in
immunoprecipitation buffer, kept on ice for 10 min, and centrifuged at
15,000 × g for 20 min to remove any insoluble
material. The supernatants were used directly for immunoprecipitation
(under native or denaturing conditions, see above) or loaded onto
10-30% glycerol gradients, prepared as described (27). Proteasome and
catalase activities were used as sedimentation markers, 20 S and 13 S
sedimentation coefficients, respectively. Fractions from the glycerol
gradients were analyzed by immunoprecipitation under native or
denaturing conditions as described above.
Characterization of Anti-C5-, Anti-C8-, and Anti-C9-specific
Antibodies--
The anti-C5 antisera failed to immunoprecipitate the
native proteasome complex (Fig. 1,
A and B) which was readily immunoprecipitated by
the anti-proteasome antisera (26). In contrast, denaturation of
proteasomes by treatment with 0.1% SDS and boiling for 2 min allowed
the anti-C5 antibodies to immunoprecipitate the 23-kDa C5 polypeptide
in a dose-dependent manner (Fig. 1). Similar experiments to
those shown in Fig. 1B blotted with other
anti-subunit-specific antibodies (anti-C2, -C8, and -C9) failed to
detect those subunits in the anti-C5 immunoprecipitates, but they were
present, as expected, in the anti-proteasome immunoprecipitates (data
not shown). Immunoblot analysis of subcellular fractions of NRK cells
with the anti-C5 antibodies (Fig. 1C) showed that the 23-kDa
C5 polypeptide is present in the nuclear, microsomal, and cytoplasmic
(S100) fractions. Similar results were obtained with CHO and HeLa
subcellular fractions (data not shown). Although the anti-C5-specific
antibodies were unable to immunoprecipitate the native proteasome, they
detected the purified proteasome complex when adsorbed to the wells of an ELISA plate (data not shown). Almost identical results to those shown in Fig. 1 were obtained with three different rabbit and four
different mice anti-C5 antisera (data not shown).
The anti-C8 and anti-C9 antibodies were characterized in a similar way.
Both antisera were unable to immunoprecipitate the native proteasome
complex, whereas they immunoprecipitated the corresponding C8 and C9
subunits after denaturation of proteasomes (Fig.
2). The anti-C8 and anti-C9 antibodies
gave similar results to those shown in Fig. 1C with
immunoblots of NRK subcellular fractions (detecting the corresponding
29-kDa polypeptides) and were also able to detect the purified
proteasome complex when adsorbed to the wells of an ELISA plate
(data not shown).
All these data permit us to draw the following conclusions with respect
to the anti-C5, anti-C8, and anti-C9 antibodies obtained: 1) the
epitopes recognized by these antibodies in their corresponding subunits
are masked in the soluble native form of proteasomes; 2) those epitopes
are clearly accessible after denaturation of the proteasome complex, as
demonstrated by immunoprecipitation of the corresponding subunits after
denaturation of proteasomes and by detection of these subunits in
Western immunoblots; and 3) similarly, these epitopes are made
accessible to recognition after adsorption of proteasomes to the wells
of an ELISA plate.
Precursor Processing of the
To analyze the processing of the pro-C5 subunit, we used pulse-chase
experiments and glycerol gradient sedimentation of cell-free extracts.
The different samples were analyzed by immunoprecipitation with
anti-proteasome, anti-C8-, or anti-C5-specific antibodies. As shown in
Fig. 4A, the anti-C5
antibodies immunoprecipitated a labeled polypeptide of 25 kDa from
total cell extracts, whose amount decreased during the chase period.
Under the same conditions, the anti-proteasome antibodies
immunoprecipitated a set of labeled polypeptides at the beginning of
the chase experiment, and both the amount of total radioactivity and
the number of immunoprecipitated polypeptides increased during the
chase period (Fig. 4B). In contrast, anti-C8 antibodies
(Fig. 4C) immunoprecipitated a set of polypeptides at the
beginning of the chase experiment, and both the amount of total
radioactivity and the number of polypeptides immunoprecipitated did not
increase during the chase period. Actually, a small initial increase
(1.4-fold) was observed, followed by a decrease (these disappeared
completely after 24 h of chase, see below). If the cell extracts
were denatured before immunoprecipitation with the anti-C5 antibodies
(Fig. 4C), we observed the disappearance of the 25-kDa
polypeptide as shown before (Fig. 4A) but now concomitantly with its conversion to a 23-kDa polypeptide. We tentatively concluded from these experiments that the precursor of the C5 subunit (25 kDa)
remains free in the cell after its synthesis and that the processed C5
subunit (23 kDa) is not free but is probably associated with some other
components of the proteasome complex.
To examine the complexes that contained the C5 precursor and its
processed form, we used glycerol gradient sedimentation of total
radiolabeled cell extracts and immunoprecipitation. All fractions from
glycerol gradients were denatured (SDS and boiling) before
immunoprecipitation with the anti-C5 antibodies. Immediately after the
pulse, the anti-C5 antisera immunoprecipitated the free non-assembled
C5 (peak at fraction 20, top of the gradient, Fig. 5A). Similar results were
obtained when glycerol gradient fractions were used directly for
immunoprecipitation without prior denaturation. After an 8-h chase, the
free C5 (Fig. 5C) was no longer detectable; and the
processed C5 subunit (23 kDa) was found to sediment with 20 S
sedimentation coefficient. Proteasome peptidase activity, detected by
hydrolysis of N-succinyl-LLVY-methylcoumarin, was observed
peaking at fraction 6, confirming the position of the active mature
proteasome at 20 S. After 3 h of chase an intermediate situation
was obtained (Fig. 5B), free C5 precursor (25 kDa) at the
top of the gradient and the processed C5 subunit (23 kDa) mainly
sedimenting at 20 S, and some at ~15 S were detected.
These results were in apparent conflict with previous research (17, 29)
that reported the presence of precursor C5 subunit in pre-proteasome
complexes. To clarify this issue we performed pre-clearing experiments.
After a 30-min pulse, radiolabeled cell extracts were pre-cleared with
an excess of anti-proteasome, anti-C8, or anti-C9 antibodies.
Afterward, the pre-cleared lysates were immunoprecipitated with the
same antibodies or with anti-C5 antibodies. Pre-clearing with any of
the three antibodies and re-immunoprecipitation with the same antibody
failed to reveal any labeled polypeptides. Anti-C8 and anti-C9
antibodies failed to immunoprecipitate any labeled polypeptides from
lysates pre-cleared with anti-proteasome antibodies. Anti-proteasome
antibodies also failed to immunoprecipitate any complex from the
anti-C8 and anti-C9 pre-cleared lysates, although some free C2 subunit
was immunoprecipitated (data not shown). Immunoprecipitations of those
pre-cleared lysates with anti-C5 antibodies demonstrate that the C5
precursor remained in the supernatants and was effectively
immunoprecipitated by the anti-C5 antibodies. Similar results were
obtained when we used pre-cleared extracts obtained after a 3-h chase
period (data not shown). A short pulse (30 min) may not have allowed
enough accumulation of labeled pro-C5, and as a consequence its
incorporation into a complex could be undetectable. To deal with this
possible criticism, we conducted pre-clearing experiments with extracts prepared from cells continuously labeled for 3 h and then chased for 24 h. Fig. 6A shows
that anti-proteasome, anti-C8, and anti-C9 antibodies immunoprecipitate
similarly labeled complexes. After a 24-h chase, only the
anti-proteasome antibodies show the immunoprecipitation of an
apparently mature proteasome complex. This complex is no longer
recognized by the anti-C8 and anti-C9 antibodies, as expected, because
these antibodies are unable to immunoprecipitate native mature
proteasomes (Fig. 2). The anti-C5 antibodies immunoprecipitated the C5
precursor under native conditions, and after a 24-h chase no labeled C5
polypeptide was immunoprecipitated (Fig. 6A). These results
were as predicted, all the C5 precursor is processed after 24 h of
chase and incorporated into mature proteasomes (not immunoprecipitated by the anti-C5 antibodies, Fig. 1). Under denaturing conditions the
anti-C5 antibodies immunoprecipitated both precursor and processed C5
subunit (Fig. 6B, pulse control (Con.)
lane) from total 3-h pulse-labeled cell extracts. The
pre-cleared lysates from the different immunoprecipitations shown in
Fig. 6A were made to 0.1% SDS (final concentration), boiled
for 2 min, and then immunoprecipitated with the anti-C5 antibodies
(Fig. 6B). Immediately after the 3-h pulse, the anti-C5
antibodies readily immunoprecipitated the precursor C5 from extracts
pre-cleared with the anti-proteasome, anti-C8, and anti-C9 antibodies,
whereas only the processed C5 is present in lysates pre-cleared with
anti-C5 antibodies (Fig. 6B). After a 24-h chase, no C5
precursor remains (Fig. 6A), and only extracts pre-cleared
with anti-proteasome antibodies show complete removal of processed C5
subunit. In contrast, the processed C5 subunit remained present and was
immunoprecipitated by the anti-C5 antibodies in those extracts that
have been pre-cleared with anti-C5, anti-C8, and anti-C9 antibodies,
respectively.
These results clearly demonstrated that most of the C5 precursor (25 kDa) is free and non-assembled in the cell, and only the processed C5
(23 kDa) is part of intermediate and mature proteasome complexes.
Processing of C5 Precursor Is Dependent on Proteasome Activity and
Takes Place in the Cytosol--
To investigate the possible dependence
of C5 processing on proteasome activity, and to study where in the cell
it takes place, we performed a series of experiments summarized by the
data presented in Figs. 7 and
8. Radiolabeled total lysates prepared
from NRK cells, treated or untreated with 5 µM
lactacystin, were used directly or pre-cleared with an excess of
anti-proteasome antibody before immunoprecipitation with the anti-C5
antibodies under denaturing conditions. Fig. 7A shows
immunoprecipitation of total cell extracts with anti-C5 antibodies
under denaturing conditions. The results show that treatment with
lactacystin prevented the processing of subunit C5 (as expected), and
the amount of total labeled C5 subunit is similar under all
experimental conditions. Fig. 7B shows that anti-proteasome
antibodies (under native conditions) immunoprecipitated an initial
complex whose formation is not affected by treatment with lactacystin
(pulse lanes, Fig. 7B). However, lactacystin
treatment prevented the incorporation of labeled subunits and the
disappearance of subunit pro-Z during the chase period (compare
chase lanes, Fig. 7B). When cell lysates were
precleared with an excess of anti-proteasome antibody and the
supernatants immunoprecipitated with anti-C5 antibodies under
denaturing conditions, the C5 precursor was readily immunoprecipitated
and diminished during the chase period in the absence of lactacystin
(Fig. 7C) as expected (see Fig. 4A). In contrast,
the C5 precursor remains unchased and not immunoprecipitated by the
anti-proteasome antibodies when the cells are incubated in the presence
of lactacystin (Fig. 7C). These data further reinforced the
conclusion that the C5 precursor (25 kDa) is a free subunit, because
its processing to the 23-kDa species is dependent on proteasome
activity, and blocking its processing prevents its incorporation into a
complex. Fig. 8 shows the results of immunoprecipitation with anti-C5
antibodies under denaturing conditions of nuclear and cytosolic
fractions of NRK cells continuously labeled for 3 h. The precursor
(25 kDa) and the processed C5 (23 kDa) subunit are clearly observed in the cytoplasmic fraction, whereas only the processed C5 subunit is
present in the nuclear fraction. Similar results were obtained in
pulse-labeled experiments of HeLa and CHO cells (data not shown). These
results showed that the processing of the C5 subunit takes place in the
cytosol, and as a consequence, only the processed C5 subunit is present
in the cell nucleus.
Mammalian proteasomes are composed of seven different The half-lives of proteasomes are between 8 and 16 days in liver (30,
31), 2 days in H6 cells (29), and 5 days in HeLa cells (32). This long
half-life of proteasomes (longer than the doubling time of cells in
culture) implies that after a radioactive pulse, labeled subunits once
incorporated into mature proteasomes would persist for long periods,
just the opposite of what is usually obtained for most cell proteins in
pulse-chase experiments. Therefore, a critical issue is whether
synthesized subunits (labeled subunits after a short pulse) also have a
long half-life like the mature proteasomes or shorter, because free
subunits or proteasome intermediates are degraded. We have measured
(densitometric scanning of autoradiograms) the radioactivity
incorporated into single subunits and proteasome complexes during
pulse-chase experiments in NRK and CHO cells. The amount of
radioactivity incorporated into Regarding the incorporation of C5 subunit into proteasome complexes
(intermediate and mature proteasomes), the current model of proteasome
assembly postulates the existence of a half-proteasome, an intermediate
containing a ring of 7 In conclusion, although a half-proteasome intermediate in proteasome
assembly may finally prove to be a correct model, the data presented
here for the C5 subunit and previous studies with the N3 subunit (13)
strongly challenge the validity of that model as postulated. The C5 and
N3 subunits may constitute exceptions, but more exceptions can be found
reviewing published data, i.e. the pro-Z subunit, that after
a pulse can be found in complexes sedimenting at 15 S and 20 S, see
Fig. 4 of Rodriguez and Castaño (17).2 Clearly more work is
needed to delineate the pathway of assembly of the eukaryotic
proteasome (36).
7
777. By
using anti-proteasome and anti-subunit-specific antibodies, we
characterized the processing and assembly of the subunit C5. The C5
precursor (25 kDa) remains as a free non-assembled polypeptide in the
cell. The conversion of the C5 precursor to mature C5 (23 kDa) occurs
concomitantly with its incorporation into 15 S proteasome intermediate
and 20 S mature proteasome complexes. This processing is dependent on proteasome activity and takes place in the cytosol. These results are
not fully compatible with the hypothesis that postulates that assembly
of proteasomes takes place via a "half-proteasome" intermediate that contains one full -ring and one full -ring of unprocessed subunit precursors.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
7777. The crystal
structure of the Thermoplasma and yeast 20 S proteasomes
(3, 4) implies the hydroxyl group of the NH2-terminal
threonine residue of the 7 identical subunits of the
Thermoplasma and 3 of the 7 distinct subunits of yeast
proteasome are responsible for catalyzing peptide bond hydrolysis.
These catalytic sites are located on the inner surface of a central
chamber formed by the two inner subunit rings.
subunits are synthesized as precursors containing a
propeptide that is cleaved off by cis- and trans-autocatalysis yielding
the mature NH2 terminus (5). Processing has been observed in Thermoplasma (6, 7), Rhodococcus (8), yeast
(9-11), and mammals (12-15). Limited self-proteolysis (cis-cleavage)
seems to be restricted to active subunits as follows: subunit
of Thermoplasma; Pre3/1, Pup1/2, and Pre2/5 of
yeast; 
/Y, Z, and X/MB1 (and the exchangeable 
-interferon
inducible subunits LMP2, MECL1, and LMP7) of mammals. The non-active
subunits in yeast (Pre4/7, Pre1/4, Pup3/3, and Prs3/6), as
shown for Pre4/7 (11), and their homologues in mammals (N3, C7-I,
C-10, and C5) are probably processed by a trans-autocatalytic reaction
(Pre4/N3 and Prs3/C5) or non-processed (Pre1/C7 and Pup-3/C-10).
peptide is dispensable and does not play any role in the assembly nor does its length or sequence, except
for the glycine at 
1 position, affect the processing (7). Similar
studies with the yeast 20 S proteasome have demonstrated that
processing of pro-Pre2p/5 (pro-Doa3) occurs when two half-proteasome precursors associate, triggering the autocatalytic removal of the
propeptides and final maturation of active sites (10). The incorporation of Pre2p/5 to the proteasome depends on its
propeptide; furthermore, this propeptide can function in
trans suggesting it serves a chaperone-like function in
proteasome biosynthesis (10). Ump1p, a short-lived chaperone, has
recently been shown to be required for the correct maturation of the
yeast 20 S proteasome, and the propeptide of Pre2p is required for the
function of Ump1p in proteasome maturation (16).
ring and a full pro-
ring with a molecular mass of 300 kDa, although not all the subunits may be present in those intermediates (15). A similar
half-proteasome intermediate is also found in yeast (16). The fast
dimerization of half-proteasomes together with the autocatalytic processing of the pro- subunits will result in the formation of
mature active proteasomes (15). Except for the study of the processing
of pro-N3 (13) and the study of the in vitro processing of
pro- (18), all published works on mammalian proteasome assembly have
used antibodies to whole proteasome complex alone or in conjunction with antibodies specific to certain subunits (C9 and C8) and to
-interferon-inducible subunits (LMP2 and LMP7). As a consequence the assembly and processing of the constitutive subunits have been
studied indirectly.
-type subunit
family and has been cloned from yeast (19), Drosophila (20),
rat (21), mouse (22, 23), and human (24). The C5 gene (PRS3/6) is
essential in yeast (19), and a single point mutation in
Drosophila C5 gene causes lethality (20). In the initial
report on C5 cDNA isolation from rat (21), it was suggested that an
NH2-terminal proteolytic processing had taken place in the
C5 subunit for its incorporation into mature proteasomes. In the
current work, we characterize the processing and assembly of the C5
subunit in mammalian systems by using C5 subunit-specific antibodies.
These antibodies immunoprecipitate the C5 precursor (25 kDa) but not
the native 20 S proteasome complex that contains the mature C5 (23 kDa). The C5 precursor remains free in the cell, and its conversion to
the 23-kDa polypeptide occurs concomitantly with its incorporation into
15 S proteasome intermediate and 20 S mature proteasomes.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Characterization of anti-C5-specific
antibodies. Rat liver proteasome was immunoprecipitated with
anti-proteasome ( -MCP) or anti-C5-specific antibodies
under native conditions or after denaturation (boiling for 2 min in the
presence of 0.1% SDS). The immunoprecipitates were analyzed by 13%
SDS-PAGE and either stained with Coomassie Blue (A) or
transferred to nitrocellulose and immunoblotted with anti-C5-specific
antibodies (B). Equivalent amounts of subcellular fractions
of NRK cells (N, nuclear; Mt, mitochondrial;
Mc, microsomes; S100, cytosol) were resolved on a 13% SDS
gel, stained with Coomassie (C), or immunoblotted with
anti-C5 antibodies (D).
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Fig. 2.
Characterization of anti-C8- and
anti-C9-specific antibodies. Rat liver proteasome was
immunoprecipitated with anti-proteasome (anti-MCP),
anti-C8-, and anti-C9-specific antibodies under native or denaturing
conditions, as indicated. The immunoprecipitates were analyzed by 13%
SDS-PAGE and either stained with Coomassie Blue (A) or
transferred to nitrocellulose and immunoblotted with anti-C9-
(B) and anti-C8 (C)-specific antibodies. Both
subunits have a molecular mass of 29 kDa.
-Subunit C5--
To begin the study
of the C5 subunit processing, we performed in vitro
transcription/translation experiments of the full-length C5 cDNA.
Fig. 3 shows the results of one of these
experiments together with immunoprecipitation of the translated
products with anti-C5 antibodies. The primary translation product of
the in vitro transcribed rat C5 mRNA (similar results
were obtained the mouse C5 mRNA, not shown) rendered a 25-kDa
protein with a mobility in SDS-PAGE identical to the purified
recombinant C5 protein (Fig. 3, compare lanes 2 and
5) and being immunoprecipitated by the anti-C5 antibodies
(Fig. 3, lane 3). These in vitro experiments demonstrated that the precursor C5 protein (pro-C5, 25 kDa) is readily
immunoprecipitated by our anti-C5 antisera.
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Fig. 3.
In vitro translation of in
vitro transcribed rat C5 mRNA. In vitro
transcribed C5 mRNA was translated in a rabbit reticulocyte lysate
and analyzed by 13% SDS-PAGE. In the same SDS-PAGE, purified rat liver
proteasome (lane 4) and purified recombinant rat C5 protein
(lane 5) were loaded and transferred to nitrocellulose for
immunoblot with anti-C5 antibodies. Lanes 1-3 show the
autoradiogram of the nitrocellulose filter. Lane 1, total
translation reaction no mRNA added; lane 2, total
translation reaction with in vitro transcribed rat C5
mRNA; lane 3, immunoprecipitation with anti-C5
antibodies of the translation products. Lanes 4 (proteasome)
and 5 (recombinant rat C5 protein) show the immunoblot of
the same nitrocellulose filter developed with the anti-C5
antibodies.
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Fig. 4.
Study of the synthesis and processing of
subunit C5 by pulse-chase experiments in NRK cells. Subconfluent
NRK cells were labeled for 30 min with 0.25 mCi/ml
[35S]methionine/cysteine and then chased with complete
medium for the times indicated at the top of each lane. At
the times indicated cells were processed for immunoprecipitation under
native (A-C) or denaturing conditions (D).
Anti-C5 antibodies (A and D), anti-proteasome
(anti-MCP, B), and anti-C8 (C) antibodies. The
figure shows the corresponding autoradiograms of 10-20% SDS-PAGE gels
used to analyze the immunoprecipitates. Exposure time was 12 h for
A, B, and D and 20 h for C (to
make more visible the decay of the immunoprecipitated complex). Note
that the proteasome polypeptide moves more clustered on this gradient
SDS-PAGE compare with continuous SDS-PAGE).
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Fig. 5.
Glycerol gradient sedimentation analysis of
pulse-chase experiments of NRK cells. NRK cells were labeled for
30 min with 0.25 mCi/ml [35S]methionine/cysteine and then
chased with complete medium for 3 and 8 h. Total extracts from
pulsed (0 h) or chased (B, 3 h; C, 8 h)
cells were loaded onto 10-30% glycerol gradients. Gradients were
fractionated and analyzed by immunoprecipitation under denaturing
conditions with anti-C5-specific antibodies, followed by 13% SDS-PAGE
and autoradiography.
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[in a new window]
Fig. 6.
C5 precursor remains soluble after
immunodepletion of radiolabeled extracts with anti-proteasome, anti-C9,
and anti-C8 antibodies. NRK cells were labeled for 3 h with
0.25 mCi/ml of [35S]methionine/cysteine and then chased
with complete medium for 24 h. A, cell extracts after
the pulse immunoprecipitated with the indicated antibodies under native
conditions. B, immunoprecipitation with anti-C5 antibodies
under denaturing conditions. Control lane corresponds to the
immunoprecipitation under denaturing conditions of the original
pulse-labeled extract. Rest of the lanes in B correspond to
the immunoprecipitation (IP) with anti-C5 antibodies of
radiolabeled extracts pre-cleared with an excess of the antibody
indicated at the top of each lane. Figure shows the
autoradioagram of the immunoprecipitates resolved by 13% SDS-PAGE.
Pro-Z ( ), C2 (), and LMP2 () proteasomal subunits are
indicated by arrows. p17 polypeptide associated with
proteasome intermediates (see also Refs. 17 and 29) may correspond to
the mammalian homologue of yeast Ump1p (16).
View larger version (45K):
[in a new window]
Fig. 7.
Precursor C5 polypeptide processing is
dependent on proteasome activity. NRK cells were labeled for 30 min with 0.25 mCi/ml [35S]methionine/cysteine and then
chased with complete medium for 3 h. Total extracts of labeled NRK
cells treated or untreated with 5 µM lactacystin were
prepared and used for immunoprecipitation. A, anti-C5
antibodies under denaturing conditions. B, anti-proteasome
antibodies (anti-MCP) under native conditions. C,
immunoprecipitation with anti-C5 antibodies under denaturing conditions
of radiolabeled extracts pre-cleared with an excess anti-proteasome
antibodies (as in Fig. 6). Panels show the autoradiograms of the
corresponding immunoprecipitates run on 13% SDS-PAGE.
View larger version (22K):
[in a new window]
Fig. 8.
Precursor C5 polypeptide processing takes
place in the cytoplasm. NRK cells were labeled for 3 h with
0.25 mCi/ml [35S]methionine/cysteine and fractionated
into nuclear and total cytoplasmic fractions, and the fractions were
subjected to immunoprecipitation with the anti-C5 antibody under
denaturing conditions. Panel show the autoradiogram of the
corresponding immunoprecipitates run on 13% SDS-PAGE.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunits
and seven different subunits that have to be assembled into an
ordered structure with the configuration 7777 (1, 2).
Proteasome subunits are synthesized as independent polypeptides encoded
by different mRNAs, and initially they should behave as free
non-assembled subunits in the cell. subunits of the mature proteasome complex seem to have the same amino acid sequence as their
primary translation product, whereas most of the subunits are
synthesized as precursors (pro- subunits) that subsequently undergo
proteolytic processing in their NH2 terminus (5). The steady state levels of free and subunits in the cell seem too
low to be detected by conventional analysis (gradient sedimentation or
gel filtration followed by immunoblot of the corresponding fractions).
Yang et al. (17) clearly demonstrated the presence of free
non-assembled subunit C9 in RMA cells after a radioactive pulse for 30 min. We have data (not shown) that demonstrate the presence of free
non-assembled subunit C2 and C9 after a 30-min pulse in NRK, CHO, and
HeLa cells. With regard to the pro- subunits, Yang et al.
(17) mentioned the detection of free pro-LMP2, and Thomson and Rivett
(13) showed the presence of free pro-N3. We have shown here the
presence of free pro-C5. Therefore, from direct analysis of cells
pulse-labeled for a short period with anti-- and anti-
subunit-specific antibodies, we can draw a tentative conclusion that
all newly synthesized and pro- subunits are initially free
non-assembled subunits.
subunits C2, C8, and C9 (data not
shown) and into subunit C5 (see Figs. 4 and 6) remained constant
after the pulse and for the next 24 h of chase. To study the fate
of proteasome intermediates, we used anti-proteasome antibodies that
recognize both intermediate and mature proteasomes and anti-C8
antibodies that only recognize proteasome intermediates. The total
amount of radioactivity immunoprecipitated by the anti-proteasome
antibodies increases during the chase, up to 5-fold with respect to the
amount of radioactivity immunoprecipitated just after the pulse (Fig.
4B) and then remained constant up to 24 h of chase
(Fig. 6). The anti-C8 antibodies immunoprecipitated a set of
polypeptides just after the radioactive pulse, and the total amount of
radioactivity immunoprecipitated increases at the beginning of the
chase (up to 1.4-fold) and then starts to decrease, disappearing
completely after 24 h of chase (Fig. 6). These results are in
perfect agreement with a cell situation in which the rate of
degradation of newly synthesized subunits and proteasome intermediates
is not very extensive together with a very efficient incorporation of
labeled subunits into mature long-lived proteasomes. This efficiency is
specifically demonstrated by the data presented for subunit C5. The
conversion of pro-C5 to the processed C5 is close to 100% (see Fig. 4,
A and B), during the chase we observed a 5-fold
decrease in pro-C5 and a 5-fold increase in the processed C5 subunit.
Moreover, all the processed C5 subunit is finally incorporated into
20 S mature proteasomes (Fig. 5) with a long half-life (
24 h, Fig.
6). A similar situation can be deduced from the data published with RMA
cells by Yang et al. (17), using anti-C9 antibodies that
recognize both proteasome intermediates and mature proteasomes, and by
Frentzel et al. (12) with antibodies to mature proteasomes
that also recognize proteasome intermediates. Our results also indicate
that the level of pre-formed proteasome intermediates in the cell lines
used in this study is low. This situation (highly efficient
incorporation of subunits into mature long-lived proteasomes and low
levels of proteasome intermediates) may not be applicable to all cell
lines, as demonstrated for H6 cells (29) where proteasome intermediates
are only 3-4-fold less abundant than mature proteasomes, and labeled
subunits are not very efficiently incorporated into mature proteasomes.
subunits, and a ring of 7 pro- subunits.
What is the evidence supporting the presence of pro-C5 in such a
half-proteasome intermediate? Nandi et al. (29) show in
their Fig. 3 that the amount of pre-C5 (labeled p1) after a pulse of 45 min is very low both in the anti-C8 and anti-proteasome
immunoprecipitates. In contrast, the mature C5 is already present in
the anti-proteasome immunoprecipitate (labeled as 1) just after the
radioactive pulse, and its amount increases during the chase (around
2-fold). Their interpretation of these results (the same explanation is
given for N3/7 and X/MB1/5) is that the pro-C5 subunit remains
for a short time in proteasome precursors. In the study of Yang
et al. (17) some pro-C5 is present in their anti-C9
immunoprecipitates after pulse (their Fig. 3D), but its
amount is very low compared with the amount of processed C5 subunit
present after 2 h of chase (their Fig. 3B). The direct
interpretation of those data is that most of the C5 subunit present in
a complex is a processed C5 subunit; however, with the experiments
presented no conclusion can be reached about the status of the pro-C5.
The data we have presented conclusively demonstrate that most of the
pro-C5 subunit is free and non-assembled, as demonstrated by its direct
immunoprecipitation after a pulse (Fig. 4), by its native molecular
weight judged by glycerol gradient sedimentation (Fig. 5), and by the
preclearing experiments with anti-proteasome, anti-C8, and anti-C9
antibodies (Fig. 6). In contrast, the 23-kDa processed subunit C5 seems
to be incorporated into a complex, immunoprecipitated by the
anti-proteasome, anti-C8, and anti-C9 antibodies and only
immunoprecipitated by the anti-C5 antibodies under denaturing
conditions (Figs. 1, 4, 5, and 6). The lack of incorporation of pro-C5
into any complex when proteasome activity is inhibited (Fig. 7)
suggests that the entrance of pro-C5 into a complex is tightly couple
with its processing. A similar situation has been described for
pre-N3/7 (13).
| |
ACKNOWLEDGEMENTS |
|---|
We thank Carmina Gutiérrez for help in handling the animals used for antibody production and Joaquín Oliva for technical assistance.
| |
FOOTNOTES |
|---|
* This work was supported in part by Comisión Interministerial de Ciencia y Tecnología Grants SAF96-0049 and SAF99-0056, Comunidad Autónoma de Madrid and Fundación Ramón Areces.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.
Recipient of a predoctoral fellowship from Ministerio de
Educación y Cultura.
§ Supported by a grant from the Comunidad Autónoma de Madrid.
¶ To whom all correspondence should be addressed: Instituto de Investigaciones Biomédicas Alberto Sols, CSIC-UAM, Facultad de Medicina de la Universidad Autónoma de Madrid, 28029 Madrid, Spain. Fax: 34-91-585-4587; E-mail: joseg.castano@uam.es.
2 S. Rodriguez-Vilariño and J. G. Castaño, unpublished data.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; MCP, multicatalytic proteinase, proteasome; ELISA, enzyme-linked immunosorbent assay; CHO, Chinese hamster ovary; NRK, normal rat kidney; PBS, phosphate-buffered saline.
| |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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