(Received for publication, December 3, 1996, and in revised form, January 14, 1997)
From the Department of Biochemistry, University of Nijmegen, 6500 HB Nijmegen, The Netherlands and Maurice E. Müller-Institut für Mikroskopische Strukturbiologie,
Biozentrum der Universität Basel,
CH-4056 Basel, Switzerland
The eukaryotic proteasome is a barrel-shaped
protease complex made up of four seven-membered rings of which the
outer and inner rings may contain up to seven different - and
-type subunits, respectively. The assembly of the eukaryotic
proteasome is not well understood. We cloned the cDNA for HsC8,
which is one of the seven known human
-type subunits, and produced
the protein in Escherichia coli. Recombinant HsC8 protein
forms a complex of about 540 kDa consisting of double ringlike
structures, each ring containing seven subunits. Such a structure has
not earlier been reported for any eukaryotic proteasome subunit, but is
similar to the complex formed by the recombinant
-subunit of the
archaebacterium Thermoplasma acidophilum (Zwickl, P.,
Kleinz, J., and Baumeister, W. (1994) Nat. Struct. Biol. 1, 765-770). The ability of HsC8 to form
-rings suggests that these
complexes may play an important role in the initiation of proteasome
assembly in eukaryotes. To test this, we used two human
-type
subunits, HsBPROS26 and HsDelta. Both these
-type subunits, either
in the proprotein or in the mature form, exist in monomers up to
tetramers. In contrast to the
- and
-subunit of T. acidophilum, coexpression of the human
-type subunits with
HsC8 does not result in the formation of proteasome-like particles,
which would be in agreement with the notion that proteasome assembly in
eukaryotes is much more complex than in archaebacteria.
The 20 S proteasome is a multicatalytic protease complex which
functions as the catalytic core of the larger 26 S proteasome. This
particle has been found in all eukaryotes analyzed to date and plays a
major role in nonlysosomal proteolysis via the selective degradation of
intracellular proteins, mostly by ubiquitin-dependent, but
also ubiquitin-independent, pathways (1-4). In the archaebacterium Thermoplasma acidophilum a particle is found with a similar
quaternary structure, containing two different subunits named and
(5). The complex consists of a stack of four rings, each containing seven subunits. The outer rings of the archaebacterial proteasome consist of
-subunits and the inner rings of
-subunits, resulting in an
7
7
7
7
complex (6, 7). In eukaryotes several different proteasomal subunits
are known, which can be divided into two classes,
-type and
-type, based on the similarity to either the
- or
-subunit of
Thermoplasma (8, 9). Seven different members of these
-
and
-type subunits may constitute, respectively, the heptameric
-
and
-rings of the eukaryotic proteasomes, each subunit residing
possibly at a defined position (10-13).
The 20 S proteasomes degrade unfolded proteins (14) and oxidized
proteins (15, 16) in vitro. Degradation is achieved by at
least five distinct catalytic activities, which can be detected with
chromogenic peptide substrates (17). Recently, the N-terminal threonine
of the -subunit of T. acidophilum was identified as the
catalytically active amino acid by x-ray crystallographic studies of
inhibitor-proteasome complexes (7) and extensive mutational analysis
(18). The N-terminal threonine results from proteolytic processing of
the N terminus, which is an essential step in the maturation of the
proteasome. In eukaryotic proteasomes not all of the
-type subunits
may be proteolytically active (18).
Little is known about the assembly of the eukaryotic proteasome
particle. Several groups have identified 13-16 S particles containing
most of the -type subunits and some
-type subunits in their
unprocessed form (19-21). These so-called "preproteasomes" are
converted into 20 S proteasomes, a process which is
translation-dependent and is accompanied by the proteolytic
processing of the
-type subunits. Expression of the T. acidophilum
-subunit in Escherichia coli revealed
that this subunit forms rings of seven subunits by itself, whereas the
pro-
-subunit forms monomers (22). Coexpression of these subunits in
E. coli resulted in the correct processing of the
-subunits and subsequent formation of functional proteasomes (22,
23). Recently, it was shown that the proteolytic processing of these
-subunits is autocatalytic and that their folding and assembly is
chaperoned by the
-subunits (24).
To study whether similar processes occur during the formation of
eukaryotic proteasomes, we analyzed the assembly properties of the
recombinant human proteasomal -type subunit HsC8 (25). We found
that, upon expression in E. coli, HsC8 forms by itself large
complexes consisting of pairs of heptameric rings, which closely
resemble the recombinant
-rings of T. acidophilum. To test whether simple proteasome-like complexes could be formed, we
coexpressed HsC8 with the
-type subunit HsBPROS26 (also called HN3)
(26) or HsDelta (also called Y) (27), but in contrast to T. acidophilum proteasome assembly, no such complexes could be
isolated.
Cloning Strategies
Mutation and PCR1 primers were purchased from Eurogentec. The NcoI and NdeI sites in the primer sequences are underlined and the mutations introduced in the proteasomal cDNAs double underlined.
The pJG4-5HsC8 clone was selected via a yeast two-hybrid screening of a
HeLa fusion cDNA library (28) with B-crystallin (29) fused to
LexA as the bait. The coding sequence of the HsC8 cDNA was
amplified from the selected clone with Pwo DNA polymerase (Boehringer Mannheim) using the oligonucleotide
5
-ATCTGCTCAATCGGCACT-3
, introducing an NcoI site containing the ATG-start codon and
the oligonucleotide BcoII, consisting of a sequence located
downstream of the polylinker of the pJG4-5 vector. The PCR product was
digested with NcoI and XhoI and ligated into the
NcoI-XhoI sites of the pET16b expression vector
(pET16bHsC8). For coexpression of HsC8 with
-type proteasomal
subunits, the XbaI-BamHI fragment of pET16bHsC8 was subcloned into the XbaI-BamHI sites of pET24d
expression vector (Novagen, pET24dHsC8). Both vectors encode the
complete HsC8 protein with only a Ser to Gly mutation at position
2.
Cloning of the HsBPROS26 cDNA and subcloning into pET3c (pET3cHsB)
has been described elsewhere (26). Recently, also longer human (HsN3)
(30) and rat (RN3) (31) BPROS26 cDNA clones have been isolated with
a putative translation start 93 nucleotides upstream of the initiation
codon of HsBPROS26. To construct the vector for expression of the
mature protein (HsBPROS26mat) in bacteria, an
NdeI site comprising a new ATG start codon 39 base pairs
downstream of the original start position the HsBPROS26 cDNA was
generated by site-directed mutagenesis using the
oligonucleotide-directed in vitro mutagenesis system
(Amersham Corp.) with the oligonucleotide 5-CAGAGGTCCAATCACCCAGAACCCC-3
as
the mutagenic primer. The NdeI-XhoI(blunt)
fragment was then cloned into the
NdeI-BamHI(blunt) sites of pET3c
(pET3cHsBmat). The HsDelta cDNA was cloned from a
cDNA library of the human cell line MV3 (kindly provided by J. van
Groningen, Nijmegen) using PCR with Taq polymerase. The primers were based on the reported cDNA sequence of human subunit Y
(EMBL data base accession no. D29012[GenBank]) (27). The 5
primer used created
also an NdeI site at the start codon:
5
-AGAATTGCGGCTACCTAACTAGCTGCT-3
. The 3
primer was: 5
-TAGGATCCAGGATTCAGGCGGGTGGTAAGGT-3
. The amplified
product was first ligated into the pCRII vector using a TA cloning kit
(Invitrogen). The NdeI-BamHI fragment was then subcloned into the NdeI-BamHI sites of pET3c
(pET3cHsD). Our HsDelta cDNA clone encodes an HsDelta protein which
has a Gln to Arg mutation at amino acid position 41 of subunit Y,
corresponding to position 7 in the mature protein (27). To produce the
vector for expression of the mature HsDelta protein
(HsDeltamat) part of the HsDelta cDNA in the pCRII
vector was reamplified with Pwo DNA polymerase using the 5
oligonucleotide:
5
-AGAATTACCACTATCATGGCCGTGCAGTTT-3
and the HsDelta 3
oligonucleotide. The generated fragment contains a
5
NdeI site with the new ATG start codon 102 bp downstream the original start site. This fragment was cloned into the
EcoRI-BamHI sites of pTZ18r. The
NdeI-BamHI fragment was subsequently subcloned into the NdeI-BamHI sites of pET3c
(pET3cHsDmat). Mutated cDNAs and cloned PCR fragments
were analyzed by sequencing with the Sequenase version 2.0 sequencing
kit (U. S. Biochemical Corp.). A summary of the expression constructs
and putative products is shown in Table I.
|
Single Expression and Coexpression of Recombinant Proteasomal Proteins
Single proteasomal subunits were expressed in the E. coli strain BL21(DE3) (32). Briefly, the pET vectors were
transformed to competent BL21(DE3) bacteria. A single colony of the
transformation plate was grown at 37 °C in 1.5 ml of LB (10 g/liter
casein, enzymatic hydrolysate (Sigma), 5 g/liter yeast extract (Life
Technologies, Inc.), 10 g/liter NaCl) containing 200 mg/liter
ampicillin (Sigma, pET3c and pET16b) or 60 mg/liter kanamycin (Merck,
pET24d) to an A550 of 0.1-0.2. This culture was
stored for maximally 20 h at 4 °C or used directly to inoculate
50 or 500 ml of LB containing the appropriate antibiotics. At an
A550 of 0.5-1.0 expression of proteasomal
proteins was induced by adding
isopropyl-1-thio--D-galactopyranoside (IPTG, Research
Organics) to a final concentration of 1 mM. Bacteria were
harvested 3 h after induction by centrifugation at 5000 × g for 15 min.
For coexpression host bacteria were cotransfected with pET24dHsC8 and a
pET3c construct containing a cDNA encoding a proteasomal -subunit. Bacteria containing both vectors were selected on LB plates containing ampicillin and kanamycin. The expression procedure was as described above except that all growth media contained ampicillin and kanamycin.
Bacterial Fractionation
Bacterial pellets were resuspended in 0.04 volume of the culture
volume in TEN300 (50 mM Tris/HCl, pH 8.0, 0.5 mM EDTA, 300 mM NaCl) and frozen at 80 °C.
The bacteria were thawed at 37 °C, lysed with chicken egg white
lysozyme (Sigma) at 0 °C, and centrifuged for 30 min at 90,000 × g at 4 °C. The supernatant (water-soluble fraction)
was used for determination of the molecular mass of the proteasomal
-type subunits.
HsC8 Purification
Recombinant HsC8 was purified from bacterial water-soluble fraction on a Sepharose Q Fast Flow column (Pharmacia Biotech Inc.). After loading the HsC8 extract, the column was washed with TEN300, and proteins were eluted with a linear gradient from 300 to 600 mM NaCl. HsC8 emerged as almost pure protein (>95%) at about 550 mM NaCl. HsC8-containing fractions were pooled and concentrated in an Amicon ultrafiltration cell using a filter (Filtron) with a cutoff of 100 kDa. For electron microscopy (see below) HsC8 protein was further purified on a Superose 6 HR 10/30 prepacked size exclusion column (Pharmacia-LKB) in 10 mM Tris/HCl, pH 7.5, followed by concentration of the HsC8 peak fractions in a microsep device (Filtron) with a cutoff of 100 kDa.
Analytical Procedures
Analytical ultracentrifugation was performed on an Optima XLA analytical ultracentrifuge (Beckman Instruments) equipped with an ultraviolet absorption system (33). The concentration of the recombinant proteasome particles was adjusted to A277 = 0.11, and the protein was analyzed at 20 °C in 50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA. Sedimentation velocity runs were performed at 52,000 rpm in a 12-mm double-sector Kel-F cell, which was filled with 0.12 ml of sample solution in one sector and the same volume of buffer measured at 230 nm in the other. Sedimentation coefficients were corrected to H2O by a standard procedure (34). Sedimentation equilibrium runs were performed at a rotor speed of 4,400 rpm in an epon-charcoal 12-mm double-sector cell filled with 1.1 ml of sample solution. The average molecular mass was determined using a linear regression computer program that adjusts the baseline absorption so as to obtain the best linear fit of ln A versus r2 (A, absorption; r, radial distance from the rotor center). A partial specific volume of 0.73 cm3/g was assumed.
The molecular mass of the proteasomal subunits and subunit complexes present in bacterial water-soluble fraction was estimated by fast performance liquid chromatography gel filtration in TEN300 on an LKB-Bromma high performance liquid chromatography system. Three different size exclusion columns were used to span a molecular mass range from 5 to 5000 kDa; Superose 6 HR 10/30, Superose 12 HR 10/30, and Superdex 75 Hiload 16/60 (Pharmacia-LKB) with separation ranges of 50-5000, 5-300, and 5-70 kDa, respectively. Gel filtration columns were calibrated with marker proteins (Pharmacia-LKB) as indicated in the figure legends. Column fractions were analyzed by SDS-PAGE (35) and either stained with Coomassie Brilliant Blue or blotted onto nitrocellulose, whereafter the specific proteasomal subunits were detected with antibodies. Antibodies used are MCP72 (culture supernatant, diluted 1:50), directed against HsC8, MCP205 (ascites, diluted 1:2000) against HsBPROS26, and MCP421 (culture supernatant, diluted 1:50) against HsDelta (36, 37), all three antibodies kindly provided by Dr. K. Hendil. Protein bands were visualized according to van Duijnhoven et al. (38).
The proteolytic activity of the subunits was tested with the synthetic
peptide substrates Suc-Leu-Leu-Val-Tyr-MCA (chymotrypsin-like), N-Bz-Phe-Val-Arg-MNA (trypsin-like) and N-Cbz-Leu-Leu-Glu-NA (peptidylglutamyl-peptide hydrolase-like). The assays were performed in
a 200-µl reaction volume containing 20 µg of crude bacterial protein, 50 mM Tris/HCl, pH 8, 150 mM NaCl, 0.1 mM EDTA, 1 mM dithioerythritol, 0.5% (v/v)
Me2SO, and 50 µM test peptide at 37 °C.
The reactions were stopped after 1 h by adding 300 µl of 1%
(w/v) SDS and 700 µl of 0.1 M sodium borate, pH 9. Released fluorogenic groups were measured in a Hitachi F-3000
spectrofluorometer at excitation and emission wavelengths,
respectively, for MNA, 333 and 413 nm; for
NA, 332 and 408 nm; and
for MCA, 339 and 439 nm.
N-terminal amino acid sequence analysis was performed at the Sequence Center of Utrecht, Institute of biomembranes, University of Utrecht.
Electron Microscopy and Digital Image Processing
Conventional Transmission Electron MicroscopyFor negative staining, a 5-µl aliquot of the HsC8 suspension diluted to about 25 µg/ml was adsorbed for 60 s to a glow-discharged carbon-coated collodium film on a 400 mesh/inch copper grid. The grid was then washed sequentially on two drops of distilled water, stained for 15 s with 0.75% uranyl formate, pH 4.25, and finally air-dried after removal of excess liquid with filter paper followed by suction with a capillary applied to the edge of the grid. Specimens were examined in a Hitachi H-8000 TEM (Hitachi Ltd.) operated at 100 kV. Electron micrographs were recorded on Kodak SO 163 (Eastman Kodak Co.) electron image film at a nominal magnification of × 52,000. Magnification calibration was performed according to Wrighly (39) using negatively stained catalase crystals.
Mass Analysis by Quantitative Scanning Transmission Electron Microscopy (STEM)For STEM mass determination of the HsC8
particles, a vacuum Generators (East Grinstead) HB-5 STEM interfaced to
a modular computer system (Tietz Video and Image Processing System
GmbH) was employed (40). For this purpose, a 5-µl drop of the HsC8 suspension diluted to about 25 µg/ml was adsorbed for 60 s to a
thin (i.e. 2.5 nm) hydrophilic carbon film which was
supported by a thick fenestrated carbon film mounted on a gold-coated
200 mesh/inch copper grid. Without negative staining, the grids were then washed sequentially on four drops of quartz bi-distilled water
with a blotting step between each wash. Finally, the specimens were
freeze-dried in the STEM's pretreatment chamber at 80 °C overnight. Low dose (i.e. ranging from 300 e
/nm2 to 1000 e
/nm2), 512 × 512-pixel elastic annular
darkfield images were recorded at 80 kV and a magnification
corresponding to a sampling distance of 0.92 nm in the specimen plane.
All microscope parameters, including the exact magnification and dose
for each image, were recorded. Evaluation of the digital electron
micrographs was carried out by the IMPSYS software package (40) running
on a VAX 3100 workstation. The mass histograms of the evaluated HsC8
particles were fitted by Gaussian curves.
From digitized electron micrographs of negatively
stained proteasome particles 64 × 64-pixel frames of 190 top
views and 110 side views were selected interactively using the SEMPER 6 image processing package (41) installed on a VAX 3100 workstation. For
both top views and side views, the respective particle images were
aligned relative to a single particle reference using cross-correlation techniques. After alignment, an intermediate reference particle image
was computed by summing up those particles yielding the highest
cross-correlation peak. This intermediate reference particle image was
then used for a second round of alignment and averaging. The final
average of the top views included 43 particles having a
cross-correlation coefficient of 0.85 with the intermediate reference
particle. To further enhance the 7-fold symmetry of the averaged top
view, it was also 7-fold symmetrized. Similarly, the final average of
the side views included 25 particles with a cross-correlation
coefficient of
0.85. To further enhance the 2-fold symmetry of the
averaged side view, it was also 2-fold symmetrized.
HsC8 was
isolated during the screening of a HeLa cDNA library with the yeast
two-hybrid system in a search for proteins interacting with the small
heat shock protein B-crystallin.2 Since
little is known about the structural properties of the eukaryotic
proteasomal subunits, we decided to study the HsC8 protein in more
detail. We therefore cloned the coding region of the HsC8 cDNA into
a pET16b expression vector (32) to produce the recombinant protein.
Upon induction with IPTG, an E. coli BL21(DE3) strain
transformed with the pET16bHsC8 construct expresses a protein of about
28 kDa (Fig. 1A). The amount of recombinant protein constituted up to 50% of total bacterial protein. The monoclonal antibody MCP72 directed to HsC8 reacts with this protein (Fig. 1B), confirming that the induced protein is HsC8.
To isolate the recombinant HsC8 protein, we extracted freeze-thawed and
lysozyme-treated bacteria expressing the HsC8 protein with buffer
containing 300 mM NaCl. The amount of recombinant protein
which remains water-soluble is over 80%. We purified the HsC8 protein
on a Sepharose Q anion exchange column, resulting in >95% pure HsC8
preparations. Peak fractions were pooled and subjected to size
exclusion chromatography on a Superose 6 column. The fractions were
analyzed by SDS-PAGE. HsC8 is eluted as a large complex in a single
homogeneous peak (Fig. 2) as has also been observed for
the -subunit of T. acidophilum (22). By comparing the
elution volume of HsC8 protein with that of calibration proteins, we
estimated the molecular mass of the HsC8 complexes to be 540 ± 30 kDa. We also analyzed the molecular mass of Sepharose Q-purified HsC8
complexes using analytical ultracentrifugation. The complex sediments
with a sedimentation coefficient s20,w
of 12.5 S, and by sedimentation equilibrium it reveals a molecular mass of 526 kDa, which is in good agreement with the gel filtration data
(see above).
Electron Microscopy
The Thermoplasma proteasomal
-subunit appeared to consist of a double ringlike structure (22). We
wondered whether the HsC8 protein forms a similar complex. Therefore we
analyzed purified HsC8 complexes by electron microscopy. Electron
micrographs of negatively stained HsC8 complexes (Fig.
3A) reveal two different images, namely
donut- or ringlike structures with a diameter of 14.3 ± 0.5 nm
and double-layered rectangular structures, 14.2 ± 0.8 nm × 12.0 ± 0.8 nm in size. They most likely represent, respectively,
the top view and side view of a particle consisting of a pair of
interacting rings. These images are very similar to electron
micrographs of the
-subunit of T. acidophilum (22, 42).
Detailed analysis of enlarged top and side views (Fig. 3B),
and correlation averaged top and side projections (Fig. 3, C
and D) revealed that, similar to the
-rings of T. acidophilum (Ta), the HsC8-rings consist of seven subunits. As can
be seen in Fig. 3C, each subunit seems to contain two
domains, a large outer domain and a smaller inner domain surrounding a
low mass density center. The center of the HsC8 rings seems to be
"plugged," contrary to Ta-
-rings, which have a small central
pore (22). The resolution of structural detail of the correlation
averaged the side view is too low to decide whether the two stacked
rings are out of register (Fig. 3D), like in Ta-
-rings
(22), or in a parallel orientation, possibly caused by different
rotational orientations of the complexes.
The complex of two heptameric HsC8 rings contains 14 polypeptides of
28.5 kDa and has a calculated mass of 400 kDa. This is significantly
smaller than the molecular mass determined with either gel filtration
or ultracentrifugation. Therefore, we also performed STEM mass
measurement of unstained freeze-dried HsC8 particles (Fig.
4A) as a third mass determination method. The STEM analysis (Fig. 4B) yielded a molecular mass of 530 ± 90 kDa, thus confirming the mass values determined by gel filtration
(i.e. 540 ± 30 kDa) and analytical ultracentrifugation
(i.e. 526 kDa). The discrepancy between the estimated
molecular mass and the calculated mass is not clear but might be caused
by a relatively large space between the two -rings (Fig.
3D).
Expression of Recombinant Proteasomal
Coexpression of the - with either the pro-
-subunit
(23) or the mature
-subunit (22) of T. acidophilum in the
host E. coli resulted in the generation of functional
proteasomes. To investigate whether such simple eukaryotic
proteasome-like particles could be produced we performed coexpression
of HsC8 with two human proteasomal
-type subunits. To our best
knowledge it is not known which
-type subunit(s) bind to HsC8.
Therefore, we chose arbitrarily the two
-type subunits HsBPROS26 and
HsDelta. At first, we expressed and characterized the assembly
properties of the
-type subunits alone in their proprotein
(HsBPROS26 and HsDelta) and mature form (HsBPROS26mat and
HsDeltamat) from constructs with, respectively, the natural
start codon or a start codon placed just upstream of the codon for the
N-terminal threonine of the processed forms.
Induction of the -type subunits encoded by the different pET
constructs resulted in the expression of polypeptides in amounts of
10-50% of total bacterial protein (Fig.
5A). Only HsDeltamat expression,
even in different E. coli BL21(DE3) host strains and using
several growth conditions, was undetectable by SDS-PAGE and Western
blotting (data not shown). The expressed proteins were separated by
SDS-PAGE and identified on Western blots with monoclonal antibodies and
compared with the homologous subunit present in human placenta
proteasomes (Fig. 5B). The bacteria expressing HsBPROS26
contain a protein which reacts with the corresponding monoclonal
antibody and is, as expected for the HsBPROS26 proprotein, larger than
the mature proteasomal homologue. HsBPROS26mat has about
the same mobility on an SDS-polyacrylamide gel as its proteasomal homologue. To ascertain that the recombinant HsBPROS26mat
indeed contains the wild-type threonine at the N terminus, the
N-terminal amino acid sequence has been determined. It appeared that
20% of HsBPROS26mat has a free N-terminal threonine, the
rest still having the translation start methionine at the N terminus.
The protein expressed from the pET-vector encoding the HsDelta
proprotein, which reacts with the anti-HsDelta monoclonal antibodies,
has almost the same mobility on an SDS-polyacrylamide gel as the mature proteasomal homologue. Only a faint band at the expected position of
the unprocessed HsDelta form can be detected in some lysates (data not
shown). The N-terminal amino acid sequence of the recombinant protein
is Ala-Val-Arg-Phe-Asp-Gly, which corresponds with the positions 5-10
in the mature protein. This means that the N terminus is
proteolytically cleaved in the bacterium or, alternatively, that the
second AUG codon, which codes for methionine at position 4 in the
mature protein, is used as a translation start site.
The HsBPROS26 proprotein and our mature-like HsDelta, both of which do
not have a threonine at the N terminus, are thus very likely
proteolytically inactive. Furthermore, HsBPROS26mat having a threonine at the N terminus, but lacking other for proteolytic activity essential amino acid residues located near the active center
(18), may also be inactive. To confirm this, we tested the proteolytic
activity in crude extracts of bacteria expressing -type subunits
using the fluorogenic test peptide substrates Suc-Leu-Leu-Val-Tyr-MCA,
Bz-Phe-Val-Arg-MNA, and Cbz-Leu-Leu-Glu-
NA. Such a test was valid to
detect the proteolytic activity of the archaebacterial
-subunit
(22). As a negative control, we used lysates of HsC8-expressing
bacteria. We could not detect a significantly elevated activity in
bacterial extracts containing any of the
-type subunits, indicating
that they indeed are proteolytic inactive.
The
molecular masses of HsBPROS26, HsBPROS26mat, and HsDelta
present in the water-soluble fraction of crude bacterial lysates were
determined using size exclusion chromatography. About 50% of the
HsBPROS26 subunit and only a small fraction (less than 10%) of the
HsBPROS26mat and mature-like HsDelta proteins are soluble
in buffer containing 300 mM NaCl. The pro--subunit and mature form of T. acidophilum behave also differently (22). Fig. 6 displays Western blots of fractions obtained with
the molecular mass determination on a size-calibrated Superose 12 column. The soluble
-type subunits have much smaller molecular
masses than the HsC8 subunit. Both the
-type HsBPROS26 and
HsBPROS26mat emerged at the monomer position of about 30 kDa. In contrast the HsDelta subunit is eluted at the tetramer position
(Fig. 6). Thus, both
-type subunits do not form ring structures upon
recombinant expression in the absence of other proteasomal
subunits.
Coexpression of HsC8 with
To test whether
the human -type subunits, like in T. acidophilum, can
assemble into proteasome-like particles with the HsC8 complex, we
coexpressed HsC8 with HsBPROS26, HsBPROS26mat or the mature-like HsDelta. The presence of both HsC8 and a
-subunit in
IPTG-induced bacteria was confirmed on Western blots (data not shown).
It appeared that HsDeltamat also could not detectably be
expressed in combination with HsC8. Coexpression of the
-type subunits with HsC8 does not result in a change in solubility of the
recombinant subunits (data not shown). The water-soluble fraction of
bacteria coexpressing HsC8 with one of the
-type subunits was
separated on a Superose 6 column and the fractions were analyzed by
Western blotting (Fig. 7). Elution profiles of HsC8
obtained with single expression of HsC8 and coexpression with the
-type subunits were similar (data not shown). Upon coexpression with HsC8 a significant amount of HsBPROS26 is present in the
HsC8-containing higher molecular mass fractions. There is, however,
still HsBPROS26 present in the monomer region, although HsC8 is present
in at least 5-fold excess (data not shown). The molecular mass of the HsC8-HsBPROS26 complex is not increased up to the size of proteasomes (which are eluted in fraction 4, data not shown), indicating that HsBPROS26 cannot form a ring structure on the HsC8 complex. The
-type subunit is also not processed into the mature form and no
proteasomal activity is present in the HsC8-HsBPROS26 fraction. Furthermore, we did not observe a change in the elution profile of
HsBPROS26mat and mature-like HsDelta on a Superose 6 column when coexpressed with HsC8, compared with single expression. Thus coexpression of the ring-forming
-type subunit HsC8 with two different
-type subunits does not result in the formation of proteasome-like complexes, suggesting that for their formation other
- and
-type subunits are needed.
In this report we present the first detailed investigation of the
assembly properties of isolated eukaryotic proteasome subunits. We show
that the recombinant eukaryotic proteasomal -type subunit HsC8 forms
ringlike structures. Accordingly, the HsC8 subunits assemble into
double-ring type structures with two rings, consisting of seven
subunits each, stacking on top of each other. Rings containing seven
subunits were also found in proteasomes of eukaryotes (43, 44) and
T. acidophilum (7). Recombinant Ta-
-subunits also form
complexes consisting of two heptameric rings. Correlation averaged top
views of both complexes appear very similar (compare Fig. 3C
with Fig. 4 in Zwickl et al. (22)). A remarkable difference between the human and archaebacterial complexes is that the
Ta-
-complex reveals a small, stain-filled hole in the center, which
is apparently far less pronounced or absent in HsC8 rings.
Unfortunately, the resolution in the side views of HsC8 double rings is
not sufficient to distinguish if the two rings are staggered, like the
Ta-
-rings, or in register relative to each other.
The observation that the eukaryotic HsC8 forms rings in the absence of
other -type proteasomal subunits was surprising, since in eukaryotic
proteasomes the
-rings may consist of seven different subunits (8,
36). Indications for this are that in purified proteasomes from human
placenta (36, 37) and yeast (45) all seven
-type subunits are found,
and that in yeast all
-type subunits except Y13 are essential for
life (see Heinemeyer et al. (8) and references therein).
Furthermore, immuno-EM localization studies of the
-type subunits
HsPROS30/HC2 and XAPC7 (10) and the
-type subunit HsBPROS26/HN3 (11)
in human placenta proteasomes indicate that these subunits were present
in two copies per proteasomal particle. The property of HsC8 to form
rings may, however, indicate that this subunit can actually occupy
several positions in
-rings of proteasome(-like) particles, which
would result in proteasome subpopulations differing in the content of
-type subunits. Such compositional variation could explain why the
monoclonal antibody p31k (46), which is directed to HsC8
(47),3 stained mainly areas in rat liver
cells that are close to the bile canaliculi, whereas a much more
general staining was obtained with a polyclonal antiserum directed to
proteasomes (48, 49). Also in C2.7 myoblasts (50), LCLC103 lung
carcinoma cells and the adherent fraction of NCI-H524 lung carcinoma
cells,4 and cells in some stages of early
embryonic development of the newt Pleurodeles waltl (51),
the p31k antibody gave different staining patterns compared with the
monoclonal antibodies directed to other
-type subunits. On the basis
of the present results it is not clear whether putative proteasomal
complexes with a high HsC8 content are assembly intermediates or
functional proteasome complexes.
Proper folding, assembly, and processing of the -subunits of
T. acidophilum is chaperoned by the
-rings (22, 24). The formation of an
-subunit ring is therefore the first event in assembly. Coexpression of the Ta-
and Ta-
-subunits in E. coli produces functional proteasomes. Since HsC8 forms similar
rings, these complexes may be important for proteasome assembly in
eukaryotes. However, coexpression of the ring-forming HsC8 and the
-type subunits HsBPROS26, HsBPROS26mat, or HsDelta did
not yield proteasome-like complexes, although a fraction of HsBPROS26
does bind to HsC8. Moreover, coexpression of
-type subunits with
HsC8 did not change their solubility either (data not shown) indicating
that HsC8 does not chaperone the proper folding of these proteins. The
three human
-type subunits tested thus apparently cannot bind
properly to HsC8 rings. This means that the HsC8 rings and Ta-
-rings
behave differently since the Ta-
-rings can assemble into
proteasome-like particles with both processed and unprocessed
Ta-
-subunits (24). A possible explanation is that eukaryotic
-subunits do interact with two different
-type subunits in the
proteasome, which define a binding pocket for a certain
-type
subunit. This is possible since the
- and
-rings are about 25°
out of register, which is approximately half a subunit (6, 7, 52).
Thus, the HsC8 ring may be an important complex for proteasome
assembly, but before
-type subunits can bind properly to the
-ring, the latter needs to consist of different
-type subunits.
In this respect it is of interest whether the ability of HsC8 to form rings by itself is a general feature of human
-type subunits. If so
the variability in the subunit composition of proteasomes may be more
extended than is believed presently. Alternatively, if only HsC8 can
form rings, these rings may be a start point for proteasome assembly.
As can also be concluded from the recent work on processing and
assembly of
-type subunits in yeast (13), our data confirm that
proteasome assembly in eukaryotes is much more complex than in
archaebacteria.
We thank Dr. K. Hendil for providing the monoclonal antibodies MCP72, MCP205, and MCP421. We also thank Dr. W. de Jong for critically reading the manuscript, and H. Smits and Dr. L. Benedetti for advice and technical assistance with the initial electron microscope studies.