From the Centro de Biología Molecular "Severo Ochoa," CSIC and Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain and § Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115
Received for publication, September 13, 2002, and in revised form, December 3, 2002
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ABSTRACT |
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We report the functional characterization
of RPN6, an essential gene from Saccharomyces
cerevisiae encoding the proteasomal subunit Rpn6p. For this
purpose, conditional mutants that are able to grow on galactose but not
on glucose were obtained. When these mutants are shifted to glucose,
Rpn6p depletion induces several specific phenotypes. First,
multiubiquitinated proteins accumulate, indicating a defect in
proteasome-mediated proteolysis. Second, mutant yeasts are arrested as
large budded cells with a single nucleus and a 2C DNA content;
in addition, the spindle pole body is duplicated, indicating a general
cell cycle defect related to the turnover of G2-cyclins
after DNA synthesis. Clb2p and Pds1p, but not Sic1p, accumulate in the
arrested cells. Depletion of Rpn6p affects both the structure and the
peptidase activity of proteasomes in the cell. These results implicate
Rpn6p function in the specific recognition of a subset of substrates
and point to a role in maintaining the correct quaternary structure of
the 26 S proteasome.
The 26 S proteasome is responsible for the
ATP-dependent degradation of short-lived regulatory
proteins involved in various biological processes such as cell cycle
and division, DNA repair, signal transduction, apoptosis, morphogenesis
of neuronal networks, metabolic regulation, and antigen presentation
(1, 2). Prior to degradation, most substrates of the proteasome are
covalently conjugated to ubiquitin
(Ub).1 Ubiquitylation
requires three enzymatic activities, the first of which activates
ubiquitin, which is then transferred to one of the
ubiquitin-conjugating enzymes. A third activity conveyed by
ubiquitin-protein isopeptide ligase is required for substrate specificity. The multiubiquitinated proteins are subsequently recognized and degraded by the 26 S proteasome (1, 3, 4).
The 26 S proteasome is a 2.4-MDa protein complex composed of two
multimeric subcomplexes, the central 20 S core particle (CP) and the 19 S regulatory particle (RP), also known as PA700. The 670-kDa CP is a
cylindrical stack of four heptameric rings whose proteolytically active
sites are sequestered within an internal chamber (5, 6). The RP is
responsible for polyUb recognition (7, 8), substrate unfolding, and
substrate translocation into the CP (9, 10), where it is degraded.
At least 17 different subunits have been identified in the RP of yeast
proteasomes (11), which is very similar to that of mammals. In
vitro the RP can be dissociated in two subcomplexes, the lid,
containing eight regulatory subunits, Rpn3, Rpns5-9, and Rpns 11 and
12, and the base, which links the RP to the CP and contains six
homologous ATPases, Rpt1-6p, as well as Rpn1p and Rpn2p. Recently,
additional proteins have been described as components of
affinity-purified proteasomes (12).
The proteasome lid subunits exhibit marked similarities to the
COP9-signalosome complex (CSN) (13), a key regulator that has recently
been demonstrated to be involved in numerous signaling pathways
(reviewed in Ref. 14). The CSN has been found in plants, mammals,
Drosophila, and Saccharomyces pombe, but
it is absent from Saccharomyces cerevisiae. The eight
subunits of the CSN are each paralogous to one of the eight subunits
that form the lid of the 26 S proteasome. The CSN and the proteasome
are evolutionary conserved particles, but they have unique structures
(15). Nevertheless, they might share a common function, because the CSN
has been reported recently (16-18) to be involved in protein
degradation. Possible cooperation between both complexes in plants has
also been reported (19). In addition, a new protein complex related to
the RP, also containing Rpn6p, has been found in Arabidopsis
seedlings and cauliflower florets (20).
Despite recent advances in the knowledge of the structure and function
of the proteasome, the enzymatic activities and specific functions
played by most regulatory subunits remain unknown. With the exception
of Rpn9p and Rpn10p, all the regulatory subunits are essential. Rpn10p
binds polyUb chain both in its free form and when incorporated into
proteasomes (21, 22). Thus, Rpn10p presumably functions as a
proteasomal polyUb protein receptor. However, other major polyUb
receptors must exist in the proteasome, because in Assigning individual roles to lid subunits should enhance our
understanding of the proteasome. To this end, a functional analysis performed with the S. cerevisiae null rpn6- Strains, Media, and Genetic Methods--
S.
cerevisiae strains used in this work are listed in Table I.
Cells were grown in synthetic dextrose minimal (SD),
synthetic complete (SC), or rich medium
with either glucose (YPD) or galactose (YPGal) as a carbon
source (37). Hydroxyurea was added to a final concentration
of 10 mg/ml and incubated for 4 h. Transformation of S. cerevisiae was carried out as described elsewhere (38). For
selection of geneticin (G418) resistance, cells were first grown for
2 h at 30 °C in liquid YPD and then spread on YPD plates containing 200 µg/ml of G418. Plasmids with URA3 as the
selection marker were cured by plating the cells onto SC plates
containing 5-fluoroorotic acid (37). Escherichia coli DH5 Gene Replacement--
Disruption of RPN6 was
performed using the short flanking homology method (40). Vector pUG6
(41) was used as a source of the geneticin resistance gene.
Hemagglutinin (HA) tagging of PDS1 (strains FS6a2 and
FSD622) and CLB2 (strains FS6a3 and FSD623) was done by
transforming either FS6a (for FS6a2) and FSD620 (for FSD622) with
plasmid pOC52 linearized with SacI and
ClaI or FS6a (for FS6a3) and FSD621 (for FSD623) with
pCLB2(HA)3 linearized with BglII. Strains used
for proteasome purification were obtained from FS6a and FSD641 by
replacing the PRE1 locus with a protein A-tagged
PRE1 (12), thus yielding the strains FS6a4 and FSD642, respectively.
Plasmid Constructions--
Relevant features of the plasmids
used in this work are shown in Table I. These were constructed as
follows. For pRC-66 and pFC7-66, the 2.1-kbp fragment containing the
Rpn6p encoding region plus 450 and 330 nucleotides from the 5' and 3'
ends, respectively, was cloned into plasmid pRS416 (42), yielding
plasmid pRC-66. The same fragment was cloned into pFL37 (43), thus
yielding pFC7-66. For pFC8-G6 and pFC9-G6, for conditional expression
of Rpn6p, the DNA encoding fragment was fused to the GAL1
promoter in centromeric plasmids pFL38 and pFL39 (44). From plasmid
pRC-66 a 1.7-kbp fragment was digested and cloned into pFLUraGP0 (45). The obtained plasmid was termed pFC8-G6. The GAL1-RPN6
fragment was also cloned in pFL39, yielding plasmid pFC9-G6. For
pFC8-GHis6, a PCR-amplified fragment of RPN6 was cloned in
plasmid pRSETC (Invitrogen), which places in the N terminus the
hexahistidine and T7 tags, yielding pRSET6. The tagged RPN6
thus generated was subcloned into pFLUraGP0 (45) to obtain pFC8-GHis6,
which harbors His6-RPN6 under the control of the
GAL1 promoter. For pFC6-6S9 and pFC7-P0S9, a 1.3-kbp PCR
fragment containing the S9 coding region amplified from a HeLa cells
cDNA library was cloned in plasmid BSP0Hs (46), yielding plasmid
pBP0S9. The 3-kbp fragment containing the S9 open reading frame flanked
by the 5' and 3' untranslated regions of RPP0 was cloned
into pFL37 (43), thus yielding pFC7-P0S9. S9 open reading frame was
also placed under the control of the RPN6 promoter by
overlapping PCRs and cloned in pFL36 plasmid (44), yielding pFC6-6S9.
The pF6M-SIC1(HA) construct was obtained by cloning
SIC1(HA) from YCp-SIC1 together with the MET25
promoter, obtained from pUG35 (kindly provided by Dr. Hegemann,
Heinrich-Heine-Universität, Düsseldorf, Germany) in plasmid
pFL46S (44).
Flow Cytometry Analysis--
DNA content was quantified with a
fluorescence-activated cell sorter (FACScalibur; BD
Biosciences). Samples containing 10,000 cells were fixed in 70%
ethanol, resuspended in 0.5 mM sodium citrate supplemented
with 0.2 mg/ml RNase, incubated for 2 h at 37 °C, and treated
with 5 mg/ml pepsin before staining with propidium iodide and sonication.
Immunological Methods--
Standard procedures were used for
immunoblotting. Tubulin was detected by using rat monoclonal antibody
YOL1/34 (Harlan Sera-Lab), polyUb-conjugated proteins were detected
with a commercially available rabbit polyclonal antibody (Affiniti),
T7-His6-tagged Rpn6p was detected with a commercially
available monoclonal antibody against the T7 tag (Novagen), and
HA-tagged proteins were detected using the monoclonal antibody 12CA5.
As a loading control in SDS-PAGE, antibodies recognizing the ribosomal
protein P0 were used (45).
Indirect immunofluorescence and 4',6-diamindine-2'-phenylindole (DAPI)
staining were performed as described elsewhere (47). Rhodamine-conjugated mouse anti-rat IgG (Biomeda Corp.) was used as a
second antibody for tubulin detection.
For protein extraction cells were resuspended in Buffer 1 (100 mM Tris-HCl, pH 7.4, 20 mM KCl, 12.5 mM MgCl2, 5 mM 2-mercaptoethanol) plus protease inhibitors. Glass beads were added, and cells were broken
with a FastPrep FP120 (Bio 101 Inc.). A total extract of 50 to 100 µg
was routinely resolved by SDS-PAGE and electroeluted onto
polyvinylidene fluoride filters (Immobilon-P; Millipore).
Scanning Electron Microscopy--
Yeast cells were resuspended
in 0.2 M sodium cacodylate, pH 7.8, and fixed in 5%
glutaraldehyde in the same buffer for 1 h at room temperature.
Cells were then spotted onto polyvinylidene fluoride membranes
(Immobilon-P; Millipore). Samples were dehydrated in a graded ethanol
series and finally in acetone. After drying, the membranes were mounted
on scanning electron microscopy stubs and then coated with gold.
Scanning electron microscopy was performed on a Philips XL30 scanning
electron microscope.
Proteasome Purification and Peptidase Activity--
Proteasome
purification was accomplished using protein A-tagged Pre1p. Details of
this procedure are provided elsewhere (12). The separation of
proteasomes by density gradient sedimentation was done centrifuging 50 mg of whole cell protein, obtained in Buffer A (50 mM
Tris-HCl, pH 8, 5 mM MgCl2, 1 mM
EDTA, and 1 mM ATP) and applied over a 10-40% continuous
glycerol gradient. The gradients were centrifuged at 25,000 rpm in an
AH-627-2A Beckman rotor for 22 h at 4 °C.
Assays of proteasome activity were done as described in Glickman
et al. (11). Briefly, 50 µg of purified proteasomes were incubated in Buffer A with 0.1 mM Suc-LLVY-AMC
(Bachem) for 30 min at 30 °C. 1% SDS was added to quench the
reaction, and released (AMC) fluorescence was read at an
excitation wavelength of 380 nm and an emission wavelength of 440 nm.
Proteasomes were resolved by non-denaturing PAGE (11), and the bands
were visualized upon incubation of the gels in 10 ml of 0.1 mM Suc-LLVY-AMC in Buffer A for 30 min at 30 °C.
Cdc28 Kinase Activity--
Yeast cultures (1010
cells) were grown to mid-log phase, harvested, washed, and resuspended
in HB buffer (25 mM MOPS, pH 7.2, 60 mM
RPN6 Is an Essential Gene Necessary for Protein
Degradation--
As a part of the yeast genome sequencing project our
group described an open reading frame named YDL097c (D2381) (33), which was later identified as a component of the proteasome RP named Rpn6p
(11). To further characterize this proteasome subunit, a cassette was
constructed containing the kanr gene flanked by
40 nucleotides from the 5' and 3' ends of RPN6, respectively, to direct the disruption of the gene in diploid strains
FY1679 and CEN.PK2. After transformation, geneticin-resistant colonies
were selected, and the correct integration of the cassette into the
genome was confirmed by PCR (data not shown). Transformants from both
genetic backgrounds were chosen for sporulation and tetrad analysis. In
all tetrads dissected, the four spores segregated in a 2:2 ratio of
viable versus non-viable spores (Fig.
1A). As expected, all the
viable spores were sensitive to geneticin, indicating that they did not
carry the disruption marker. This result indicates that Rpn6p function
is essential for either vegetative growth or spore germination.
Heterozygous RPN6-disrupted diploids (FS6D6) were
transformed with the centromeric plasmid pFC8-G6, which directs the
expression of RPN6 under the control of the GAL1
promoter, and allowed to sporulate and then tetrads were dissected. As
expected from the previous results, only two colonies per tetrad grew
on glucose whereas all four spores formed colonies on galactose plates
(data not shown). Geneticin-resistant haploid cells
rpn6- Human Proteasomal Protein S9 Partially Rescues the Effect of Rpn6p
Depletion--
Rpn6p was first suggested to be a component of the
yeast proteasome based on a 42% identity to human proteasome subunit
S9 (34). To determine whether this sequence similarity extends to its
function, the ability of the human S9-encoding cDNA to complement
the rpn6- Cell Cycle-related Defects of Conditional Null Mutants
rpn6-
These results demonstrated clearly that cells lacking Rpn6p were
arrested in the cell cycle after DNA synthesis, in G2/M. As
progression of the cell cycle from metaphase is directly related to the
inhibition of the Cdc28 kinase activity (48), a preliminary study of
the variations in the level of this activity was approached. For this
purpose, whole cell extracts from FSD60 mutant strain maintained on
glucose were assayed to measure their histone H1 phosphorylating
activity. As shown in Fig. 4D, this activity progressively increased, reaching a maximum after 9 to 12 h.
The results aforementioned suggested that Rpn6p is essential for
progression from S/M to G1. This transition is driven by the anaphase-promoting complex/cyclosome (APC/C), which is switched on
at the metaphase/anaphase boundary to trigger the ubiquitination and
proteolysis of sister chromosome cohesion factors and mitotic cyclins
(49). The stability of two critical APC targets, Clb2p and Pds1p, was
therefore examined. Wild type and rpn6-
The homogeneous phenotype described in the experiments shown above
implicated Rpn6p in the degradation of two substrates ubiquitinated by
APC/C. To study the role of Rpn6p in the degradation of substrates ubiquitinated by a different ubiquitin ligase, variations in the level
of Sic1p were measured. Degradation of Sic1p, an inhibitor of
Clb/Cdc28p kinase activity, is necessary for cells to enter S phase
(48). Ubiquitination of Sic1p by the ubiquitin-protein isopeptide
ligase SCFCdc4
(Skp-cullin-F-box protein) during
S/M triggers its degradation by the 26 S proteasome (51, 52). To
determine whether Sic1p proteolysis depends on the presence of Rpn6p in
the proteasome, we examined the stability of Sic1p in
rpn6- Structure and Peptidase Activity of Rpn6p-depleted
Proteasomes--
Rpn6p harbors a structural motif, the PCI
domain (53), also present in other five proteasome subunits and in
components of distinct multiprotein complexes such as COP9 and eIF3
(13). It has been suggested that the PCI domain could serve as a
structural scaffold helping the subunits to assemble in these
multimeric complexes (54, 55). If this was the case,
rpn6-
Regarding the defect in peptidase activity, in vitro
activity assays were performed to test the ability of mutant
proteasomes to hydrolyze the fluorogenic peptide Suc-LLVY-AMC.
Confirming the results described above, the in vitro
peptidase activity of proteasomes lacking Rpn6p was drastically reduced
compared with wild type (Fig.
7A). As it was one possibility
that this lower peptidase activity was because of a lesser amount of
proteasomes produced when Rpn6p is depleted, the purified proteasomes
were pre-incubated in the absence of ATP to release their CPs. As shown in Fig. 7A, under this condition the basal peptidase
activity of wild type and mutant proteasomes was similar, thus
indicating that the absence of Rpn6p does not affect the CP.
Furthermore, under dissociation conditions, together with a mild SDS
treatment, which also stimulates the peptidase activity of the yeast CP
(56), there is a 50-fold stimulation of the mutant CP activity (Fig. 7B). This stimulation, exceeding that of wild type CP
itself, confirmed that the presence of incomplete RPs in
rpn6- Composition of Proteasomes from rpn6- In this report an analysis of the function of Rpn6p, a proteasome
RP subunit (11), is presented. RPN6 gene disruption in two
different genetic backgrounds demonstrated that this protein is
essential for vegetative growth, in agreement with results reported
previously (35). As expected for a proteasome subunit, ubiquitin-dependent degradation is impaired in a
conditional rpn6- Proteasome subunits are well conserved in all eukaryotic organisms, and
the overall composition of the holocomplex is nearly the same from
yeast to human (57). However, when comparing individual human and yeast
proteins, differences among them can be observed. Although the identity
among the homologous ATPases is about 70%, that of most of the
non-ATPase components is decreased to about 40% (11). Rpn6p shows a
42% identity to human S9 subunit. Complementation of the
rpn6- To further analyze the function of Rpn6p,
galactose-dependent conditional null rpn6- Interestingly, rpn6- Proteasomes assembled in the rpn6- It has been reported recently (23) that enhancement of the peptidase
activity of yeast CP observed upon RP binding is because of the opening
of a channel into the CP, allowing entrance of protein substrates. In
fact, it has been shown that one of the six ATPases present in the base
of the RP, Rpt2p, is indeed controlling the gating of this channel
(24). Another ATPase from the base, Rpt5p, appears to mediate polyUb
chain binding (8). The RP particle is thus not only necessary for
substrate recognition, unfolding and translocation, but also for
opening the door for substrates into the proteolytic active chamber
within the CP. However, the function of the lid is not yet completely
clear. One recently reported function of the lid is to mediate
deubiquitination of proteins destined to be degraded by the proteasome
(31, 32). A zinc-binding site within Rpn11p is necessary for the
deubiquitination of Sic1p, and proteasomes containing amino acid
substitutions within predicted meta-chelating residues of Rpn11p are
unable to either deubiquitinate or degrade this substrate in
vitro, suggesting that one function of the lid is to serve as a
specialized isopeptidase that couples deubiquitination and degradation
of substrates. Nonetheless, it remains unclear whether there are other
deubiquitinating enzymes involved in the deconjugation of Ub from
proteasome substrates. In fact, rpn6- Here is shown additional evidence that the RP lid is directly involved
in the enzymatic activity of the 26 S proteasome. Although under
dissociating conditions, i.e. the RP is released from the CP, both wild type and CPs isolated from the rpn6- The above data are limited to Rpn6p function in 26 S-dependent degradation. However, it would be interesting
to find out whether Rpn6p is implicated in the novel activities
described recently for the RP as transcription elongation (67) or
nucleotide excision repair (68, 69) and its relevance in a distinct
function in the lid context, which would account for the similarities
found with the COP9-signalosome complex.
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ABSTRACT
INTRODUCTION
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DISCUSSION
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rpn10
strains only short-lived proteins, degraded by the Ub-fusion
degradation pathway, are known to be stabilized (21). Rpn10p is also
important for correct association of lid and base (13). The base is
sufficient to activate CP proteolysis of certain non-Ub proteins,
governing their entry into the CP (11). It is responsible for the
unfolding function of the RP (9, 10) and functions as a gating device
for the CP channel; this last role played primarily by Rpt2p (23, 24).
Recently, a new mediator of ubiquitin chain recognition has been
described, Rpt5p, one of the ATPase subunits from the base (8). Another subunit of the base, Rpn1p, acts as a receptor for Rad23p (22), a
protein that might deliver proteins to the proteasome for degradation (25). Less is known about the role played by lid subunits during protein degradation. However, the lid is required for the degradation of polyUb-conjugated substrates, at least in vitro (13).
Among the essential lid proteins, only Rpn3p, Rpn11p, and Rpn12p have been further characterized (26-28). As it is the case for the 20 S and
ATPase components, they have been found to be essential for cell cycle
progression, demonstrating the important role of Ub-mediated
proteolysis in the control of the cell cycle (29, 30). Recently Rpn11p
has been described as a metalloisopeptidase involved in the
deubiquitination of proteasome substrates (31, 32).
1
conditional mutant is presented in this report. Protein Rpn6p was first
described as an open reading frame with unknown function, found during
the systematic sequencing of the yeast S. cerevisiae genome
(33). It was later identified as the human S9 homologue (34, 35) and as
a component of the S. cerevisiae RP (11). Additional characterization of this subunit as a proteasome component that interacts with CSN has been carried out in Arabidopsis and
Drosophila (19, 20, 36). Very recently, it has been reported
that Rpn6p is essential for Drosophila development (36). The
data presented in this report suggest that Rpn6p plays a key role in
the lid of the yeast RP. It is important for maintaining the correct
assembly of 26 S proteasomes and facilitates the proper accommodation
of other lid subunits. Moreover, Rpn6p subunit is necessary for the complete activation of 26 S upon binding of the RP to the CP. In
addition, Rpn6p function is essentially required during mitosis.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was used for cloning steps and was grown in LB medium. Standard DNA
cloning and manipulation were performed according to well established techniques (39). PCR amplifications were made using the high fidelity
Pfu (Stratagene) or Pwo (Roche Diagnostics) DNA
polymerases.
Yeast strains and plasmids used in this work
-glycerophosphate, 15 mM
p-nitrophenylphosphate, 15 mM MgCl2,
15 mM EGTA, 1 mM dithiothreitol, 0.1 mM sodium vanadate, 1% Triton X-100, 1 mM
phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, 40 µg/ml
aprotinin) and broken with 2 volumes of acid-washed glass beads. 60 µg of extract was incubated for 20 min at 37 °C in the presence of
H1 histone (0.5 mg/ml) and [
-32P]ATP (100 µM, 100 cpm/pmol). Reaction was stopped by the addition of one volume of 2× sample buffer, and samples were resolved by 10%
SDS-PAGE.
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ABSTRACT
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DISCUSSION
REFERENCES
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Fig. 1.
RPN6 is an essential gene.
A, tetrad analysis of diploid CEN.PK2 cells containing one
disrupted copy of RPN6. Spores were grown on a glucose plate
at 30 °C for 3 days. B, conditional null
rpn6- 1 mutants do not grow on glucose. Wild type cells
(FS6a) or conditional rpn6-
1 mutants expressing Rpn6p
(FSD61) or Rpn6p(His)6 (FSD641) under
the GAL1 promoter were grown on galactose to mid-exponential
phase and then plated onto YPGal or YPD and incubated at 30 °C for 3 days. C, strains FSD641 (His6Rpn6p) or FS6a
(Rpn6p) were grown on galactose to late exponential phase
and then transferred to glucose for 24 h. At the times indicated,
samples of the cultures were harvested and lysed. 100 µg of total
proteins were analyzed by immunoblot with antibodies recognizing the T7
tag. The immunodetection of ribosomal protein P0 was done as a loading
control. D, Ub proteins accumulate in rpn6-
1
mutants. Whole cell extracts from strains FSD61 (rpn6-
1)
or FS6a (RPN6) grown as explained for C were
subjected to SDS-PAGE and immunoblotting with anti-Ub antibodies.
1 from strain FSD61 stopped cell division after
prolonged incubation in the restrictive glucose-containing medium (Fig.
1B), allowing further study of the function of Rpn6p.
Initially, the depletion of Rpn6p on glucose in these mutants was
studied. A His6-tagged version of Rpn6p was cloned in the
same centromeric plasmid under the control of the GAL1
promoter as explained above, and the same procedure was followed to
obtain strain FSD641, which conditionally expresses
His6-Rpn6p on galactose. The His6-tagged Rpn6p
fully complemented the absence of the endogenous Rpn6p on galactose, as
no differences in the growth rate were observed (Fig. 1B). After transferring the cells to restrictive conditions, growth was
monitored, and cell samples were collected and analyzed by immunoblotting. As shown in Fig. 1C, His6-Rpn6p
progressively disappears once the cells are transferred to glucose,
being undetectable after 9 to 12 h. In parallel, polyUb proteins
began to accumulate after 6 h under these restrictive conditions,
reaching the maximal amount after 9 to 12 h (Fig.
1D).
1 mutation in S. cerevisiae was
tested. Centromeric plasmids pFC6-6S9 and pFC7-P0S9, expressing,
respectively, S9 under the control of either the RPN6 or the
ribosomal protein P0 promoters, were used to transform
rpn6-
1 mutant cells, and their ability to grow on glucose
was tested. Fig. 2A shows that S9 gene expressed under the control of the RPN6 promoter
only partially restores growth on glucose (FSD631). In contrast, when overexpressed under the control of a stronger promoter, such as that of
the RPP0 gene (FSD611), the growth defect was suppressed almost completely. However, as shown in Fig. 2B, even when
highly expressed, human S9 did not completely abolish the accumulation of polyUb proteins.
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Fig. 2.
The homologous human S9 protein complements
the rpn6- 1 mutation. A, conditional
strains FSD63 or FSD61 were transformed with plasmids expressing the
human S9 protein under the control of the RPN6 promoter
(FSD631) or RPP0 promoter (FSD611) or
with a plasmid expressing the native Rpn6p (FSD64).
Exponentially growing cultures of these strains and the wild type FS6d
were diluted, and 105 cells and four successive 10-fold
dilutions were spotted on YPGal, YPD, or SD-5-fluoroorotic acid plates
and incubated at 30 °C for 2, 3, and 6 days, respectively.
B, effect of S9 expression on the accumulation of multiUb
proteins. The same cultures were grown in liquid YPD for 12 h, and
50 µg of total protein was subjected to SDS-PAGE and immunoblotting
with anti-Ub antibodies. 1, FS6d; 2, FSD61;
3, FSD64; 4, FSD631; 5, FSD611.
1--
We characterized the terminal phenotypes of diploid
FSD60 and haploid FSD61 cells after shifting from galactose to glucose. The morphology of the cells was followed by Nomarski optics, scanning electron microscopy, and fluorescence microscopy. After 9 h on glucose, a homogeneous culture of large budded cells was observed (Fig.
3, A-D). Mother and daughter
cells failed to separate (Fig. 3E), and they contained only
one nucleus, which was located close to or in the bud neck in most
cases, as shown by DAPI staining (Fig. 3F). Moreover,
tubulin staining revealed the duplication of the spindle pole body,
with the presence of a short intranuclear mitotic spindle in the
arrested cells (Fig. 3G). To further analyze whether DNA
duplication proceeded normally in rpn6-
1 cells, the DNA
content of mutant arrested cells was measured. Fig.
4, A and B shows
the cytometry profiles of asynchronous wild type and
rpn6-
1 mutant cultures growing on glucose. On galactose
the two cultures showed a similar profile at all time points, with 60%
of the cells having a 2C content when growing in mid-log phase. A 2C
DNA content was observed in 80% of the mutant cells after 6 h on
glucose and in almost 100% after 9 h. However, treatment with
hydroxyurea revealed that only 30% of the cells had actually completed
DNA replication after 6 h in the restrictive condition, whereas
all of them were arrested after 9 h (Fig. 4C).
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Fig. 3.
Cytological analysis of rpn6- 1
cells under restrictive conditions. A-D,
microscopic analysis with Nomarski optics of cells maintained for
9 h in glucose. A, FSD61 (rpn6-
1
haploid); B, FS6a (wild type RPN6 haploid);
C, FSD60 (rpn6-
1 diploid); D,
FY1679 (wild type RPN6 diploid). E, scanning
electron micrographs (SEM) of FSD60 cells after 12 h
under restrictive conditions. F and G, FSD60
cells were stained with DAPI (F) and with anti-tubulin
antibodies using rhodamine-conjugated secondary antibodies
(IF) (G). The scale bar in all
panels is 2 µm.
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Fig. 4.
rpn6- 1 mutants arrest in
G2/M and accumulate Cdc28 kinase activity. Wild type
FS6a (A) or mutant rpn6-
1 FSD61 (B)
strains were grown on galactose to mid-exponential phase, and samples
were withdrawn at the indicated times after being transferred to
glucose, fixed and stained with propidium iodide, and analyzed by flow
cytometry. C, duplicated samples from FSD61 (B)
were incubated for 4 h in the presence of hydroxyurea prior to
fixation and propidium iodide staining. D, histone H1 kinase
activity was assayed in vitro from 60 µg of total protein
extracts obtained from strain FSD61 at the indicated times after
transfer to glucose.
1 conditional strains, both tagged with a triple HA epitope at their CLB2
or PDS1 loci, were shifted from galactose to glucose and
harvested at different time points. The abundance of Clb2p or Pds1p was estimated by immunoblotting against HA. As shown in Fig.
5A, Clb2p was almost
undetectable in the wild type cells after 9 to 12 h under
restrictive conditions, whereas there was a striking accumulation of
this cyclin in the mutant cells, reflecting a significant
stabilization. As shown in Fig. 5B, Pds1(HA)3
levels similarly increased in the mutant cells under the same
conditions. Accumulation of Pds1p, however, was not as obvious as that
of Clb2p. To further confirm this observation, PDS1 was
deleted in an rpn6-
1 conditional strain. Because it is
well established that Pds1p is not essential for growth at 25 °C
(50), a pds1-
1 rpn6-
1 conditional strain behaved as
wild type under permissive conditions (galactose at 30 °C), and the
double mutant was expected to avoid the metaphase arrest because of
Pds1p accumulation under restrictive conditions. In fact, this mutant
strain, when shifted to glucose, arrested as large budded cells in
which more than 50% of the population had progressed to telophase,
having segregated the nuclei to the emerging buds (data not shown).
This result clearly confirmed that the metaphase arrest phenotype in
the rpn6-
1 mutant cells is because of its inability to
degrade Pds1p.
View larger version (53K):
[in a new window]
Fig. 5.
rpn6- 1 cells are defective in
APC-dependent degradation. Wild type (RPN6)
and rpn6-
1 mutant cells, expressing either HA-tagged
Clb2p (FS6a3 and FSD623), Pds1p (FS6a2 and FSD622), or Sic1p (FS6d1 and
FS632), were grown on galactose and then transferred to glucose medium.
Cells growing on galactose (GAL), or after transferring to
glucose (at the indicated times), were recovered, and 50 µg of whole
lysates were analyzed by immunoblot with an anti-HA antibody. Ribosomal
protein P0, immunodetected with a polyclonal antibody, was used as a
loading control. Extract from a wild type strain expressing the
untagged proteins was used as a negative control.
1 cells. Wild type and rpn6-
1 mutant
cells were transformed with a plasmid carrying an HA-tagged copy of
SIC1 under the control of the MET25 promoter, yielding strains FS6d1 and FSD632, respectively. These strains were
grown under conditions allowing the expression of HA-tagged Sic1p and
harvested for further analysis (Fig. 5C). Immunoblot analysis using an anti-HA-specific antibody revealed that Sic1p levels
were comparable in wild type and mutant cells, indicating that Sic1p
turnover is not compromised in rpn6-
1 cells and
suggesting that Rpn6p is not involved in G1 degradation.
1 mutants should be unable to properly assemble 26 S
proteasome particles. To study the assembly of the 26 S proteasomes,
whole cell lysates from wild type and mutant strains, incubated for
12 h in glucose, were analyzed directly by non-denaturing gel
electrophoresis. Proteasome particle bands were visualized using a
fluorogenic peptide overlay assay to detect peptidase activity.
Dramatic differences were observed in the pattern of migration of
proteasomes from the rpn6-
1 strain when compared with
wild type (Fig. 6A). To further study their assembly state, proteasomes from wild type and
rpn6-
1 conditional strains grown under restrictive
conditions were purified by affinity chromatography, and equal amounts
of proteins were subjected to non-denaturing gel electrophoresis. Most
proteasomes lacking Rpn6p migrated faster than wild type (Fig.
6B). Although equal amounts of proteins were loaded to each lane, the intensity of the band corresponding to mutant proteasomes was
diminished strongly, suggesting a defect in assembly and/or peptidase
activity. A similar result was obtained when proteasome preparations
were analyzed by density gradient centrifugation. Both in wild type and
mutant strain, the proteasome peptidase activity fractionated
predominantly as a single peak. Nonetheless, the activity peak
displayed by the rpn6-
1 strain moved to a lighter fraction (Fig. 6C), confirming that the mutant proteasomes
were not complete 26 S particles. To check the presence of proteasomes in the gradient, the fractions were subjected to immunoblot against Rpn12p and Rpt6p, which are, respectively, located in the lid and the
base of the RP. As shown in Fig. 6D, the amount of Rpn12p in
Rpn6p-depleted proteasomes was reduced drastically compared with wild
type, whereas there were no differences in Rpt6p levels, supporting our
previous hypothesis that the rpn6-
1 strain assembles incomplete 26 S particles.
View larger version (45K):
[in a new window]
Fig. 6.
Proteasome structure is altered in
rpn6- 1 mutant cells. Wild type RPN6
(FS6a4) and mutant rpn6-
1 (FSD642) strains were grown
overnight on liquid YPGal medium and transferred to glucose for 12 h. A, whole cell lysates were subjected to non-denaturing
PAGE. Proteasome bands were visualized in situ by their
peptidase activity against the fluorogenic substrate Suc-LLVY-AMC.
B, proteasomes from wild type (RPN6) and mutant
(rpn6-
1) strains, purified either by conventional
chromatography (Convent.) (11) or by affinity chromatography
(12), were separated by non-denaturing PAGE. The first two
lanes correspond to wild type holoenzyme (Holo.) and
CP, kindly provided by M. Schmidt, and used as a control. *, this
band corresponds to CP bound to a regulator. (M. Schmidt,
personal communication.) C, whole cell extracts
from FS6a (RPN6) and FSD61 (rpn6-
1) strains
incubated in glucose for 12 h were fractionated by density
gradient centrifugation. 50 µl from each 1-ml fraction were assayed
for their hydrolyzing activity. D, 50 µl from the
indicated fractions were subjected to SDS-PAGE and immunoblotted with
antibodies against the RP subunits Rpt6p and Rpn12p, kindly provided by
Carl Mann and Akio Toh-e, respectively.
1 strain has an inhibitory effect on the peptidase
activity, highlighting the Rpn6p relevance to maintain both the proper
assembly of 26 S proteasomes and their full peptidase activity.
View larger version (24K):
[in a new window]
Fig. 7.
Proteasome activity in rpn6- 1
mutant cells. A, the same wild type and
rpn6-
1 proteasomes purified by affinity chromatography
shown in Fig. 6B were tested for their ability to hydrolyze
the substrate Suc-LLVY-AMC. Equal amounts of proteasomes were incubated
in the presence (+ATP) or absence (
ATP) of ATP
to dissociate CP and RP. B, peptidase activity of wild type
and mutant CP before (
SDS) and after (+SDS) the
activation of the basal activity with SDS. The CPs from wild type and
rpn6-
1 proteasomes were released upon incubation of the
proteasome samples in Buffer A without ATP and 500 mM NaCl
for 30 min at 30 °C.
1 Strain--
The results
shown above indicated that Rpn6p depletion was responsible for
proteasome disassembly in the mutated strain, suggesting also that
proteasomes assembled in the absence of Rpn6p might lack other
subunits. To study the composition of Rpn6p-depleted proteasomes, the
holoenzymes were purified from a wild type and an rpn6-
1
strain, both expressing the CP subunit Pre1p fused to the
TeV-ProA tag. As the main differences were expected in the
composition of RP, these complexes were also purified separately to
avoid CP subunits. Holoenzymes and RP purified from the mutant strain
grown under restrictive conditions were subjected to SDS-PAGE analysis.
As there are not commercially available antibodies to detect the
distinct RPs, comparison of subunit composition was based on the
published pattern of migration of CP and RP subunits in an SDS-PAGE gel
(11, 12). Analysis of the subunits present in wild type and mutant
strains revealed the lack of most RP subunits (Fig.
8) except for Rpn1p and Rpn2p, clearly
present in Rpn6p-depleted proteasomes, together with the ATPases, all
of them components of the proteasomal base.
View larger version (71K):
[in a new window]
Fig. 8.
Subunit composition of Rpn6p-depleted
proteasomes. A, wild type (RPN6) and mutant
(rpn6- 1) 26 S proteasomes, purified by affinity
chromatography using the Pre1-TeV-ProA fusion from strains FS6a4 and
FSD642 incubated for 12 h under restrictive conditions, were
subjected to SDS-PAGE and stained with Coomassie Blue. B,
RPs from the same strains eluted with high salt concentration.
Components of the RP are labeled based on the published data (11,
12).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 mutant, which accumulates
multiubiquitinated proteins under restrictive conditions.
1 mutation with the homologous human S9 cDNA was only possible when overexpressed under the control of the strong ribosomal protein P0 promoter but not under the endogenous
RPN6 promoter. Upon overexpression of S9, the growth
inhibition because of Rpn6p absence was relieved completely. However,
some multiubiquitinated proteins remained accumulated. A possible
explanation is that, despite the conservation of the sequence of this
subunit, human S9 is not able to recognize certain yeast substrates. In
fact, whereas hS13 can complement Rpn11p absence (27), hS14 does not complement the Rpn12p absence (28), and hS7 only does so when overexpressed in an Rpt1p mutant strain (58), even though they have a
75% identity. This led us to hypothesize that the function of the RP
subunits is species-specific, reflecting a specific recognition of
certain substrates restricted to each organism. Supporting this idea,
it has been described recently (59) that S9 interacts specifically with
NF-
B2 precursor protein p100. This interaction requires p100
ubiquitination and mediates its processing, which is required for
generating the p52 NF-
B subunit.
1
mutants were constructed. After prolonged incubation of the conditional
strain under restrictive conditions (9-12 h on glucose), when no Rpn6p
is present, rpn6-
1 cells are blocked in metaphase. They
arrest with a uniform terminal phenotype, large budded cells with a 2C
DNA content and an undivided nucleus located near the neck. In
addition, tubulin staining reveals that the arrested cells have
duplicated the spindle pole body but were arrested with a short
intranuclear spindle, which correlates with the absence of chromosome
segregation. This metaphase block was confirmed by the inability of
rpn6-
1 cells to degrade two APC/C substrates, Clb2p and
Pds1p, as a consequence of a failure in the APC/C-dependent
degradation by the 26 S proteasome. Pds1p (securin) is a key regulatory
protein responsible not only for the onset of anaphase but also
implicated in the exit from mitosis (60, 61). Pds1p is bound to Esp1
(separase), and this association helps to correctly locate Esp1 in
nucleus and spindle (62). At the onset of anaphase, the Ub ligase APC/C
targets Pds1p for 26 S-dependent degradation, and separase
is thus released, resulting in the elimination of cohesion among sister
chromatids and in spindle movement (60). It is known that
pds1 mutant cells are viable (50). Moreover, they do not
liberate chromatid sisters prematurely, and separase still localizes
properly and cleaves cohesin in a regulated fashion (63)
suggesting that Pds1p degradation is not the sole requirement for
progression through mitosis, and an alternate mechanism to regulate
Esp1 activity should exist. Indeed, rpn6-
1 mutants
accumulate polyUb proteins, indicating a failure to degrade other
substrate proteins and probably among them those accounting for the
mechanism that cooperatively with Pds1p regulate faithful chromosome
segregation during cell division.
1 mutants retain the ability to
degrade other substrates, such as Sic1p, which means that
Rpn6p-depleted proteasomes maintain partial activity. This feature is
not unique for rpn6-
1 mutants but has been also described
for other proteasome subunits. Among the RP proteins, it has been shown
that depletion of ATPases Rpt1p and Rpt6p (58), Rpt4p (64) and
non-ATPases Rpn3p (26), Rpn9p (65), Rpn11p (27), and Rpn12p (28)
produce similar phenotypes. However, even among the proteasome mutants that are arrested in G2/M, differences in the
above-mentioned phenotypes are observed. Available data in the
literature report that although rpt1, rpt6,
rpn3, and rpn9 mutants duplicate the spindle pole
body and have a short intranuclear spindle (26, 58, 65),
rpt4 mutants fail to duplicate it (64). With respect to
Cdc28/Clb2p kinase activity, it is absent in rpn12 mutants (28) but present in rpt1, rpt6, and
rpn3 (26, 58). The phenotype displayed by
rpn6-
1 mutants appears to match more closely that of the
ATPases rpt1 and rpt6 and the non-ATPases
rpn3 and rpn9. This suggests that among
proteasome regulatory subunits a specialization in substrate
recognition exists that might represent an additional mechanism of the
spatial- and temporal-regulated degradation performed by the
ubiquitin-proteasome pathway. A physical association among these
subunits might explain the parallels in their substrate recognition properties.
1 strain are clearly
different in size and subunit composition from wild type 26 S
proteasomes. Moreover, most of the lid RP subunits, if not completely
absent, are not incorporated in mutant proteasomes as they are in the wild type. We may then conclude that Rpn6p is important to maintain the
proteasomal lid bound to the base in vivo. A similar role has been described for Rpn10p, as in the absence of this protein purified proteasomes do not contain the lid subunits (13). However, contrary to rpn6-
1 mutants,
rpn10 cells are
viable. As most lid subunits are essential, the composition of purified
proteasomes from
rpn10 cells cannot reflect the situation
in vivo, indicating that, in the absence of Rpn10p, lid
subunits can interact with the base, assembling functional proteasomes,
as revealed by native gel electrophoresis (13). This is not the case
for rpn6-
1 cells, as demonstrated by density gradient
sedimentation and native gel electrophoresis analysis, highlighting the
importance of Rpn6p for the correct assembly of the lid into the 26 S
proteasome. While this manuscript was under revision, it was reported
that proteasomes purified from an mpr1-1 strain with a
frameshift in RPN11 also lack lid subunits (31). Taken
together with our results, it can be expected that the deletion of
additional RPN genes will also render proteasomes inactive
as a consequence of the improper assembly of the RP. Within this
context, the presence of a PCI motif in the C terminus of six of the
eight lid subunits may be significant. This PCI domain has been
described as a structural scaffold that may help the assembly of
subunits into multimeric complexes (54, 55). In fact, the presence of
an intact PCI domain is essential for Rpn6p function as the expression
of C-terminally truncated Rpn6p derivatives is not able to restore the
growth of rpn6-
1 mutants under non-permissive
conditions.2
1 cells are still
able to degrade Sic1p in vivo, suggesting that the function
of Rpn11p might be partially redundant with other enzymes in the cell
(66).
1
strain showed comparable peptidase activity; the proteolytic CP appears
to be inhibited by the binding of the incomplete Rpn6p-depleted RP. The
mutant proteasomes, however, maintain the ATPase activity (data not
shown), suggesting that the ATPases are not affected by the absence of
Rpn6p. Thus, in principle, the incomplete RP should be able to bind to
CP and mediate its activation through Rpt2p. Nevertheless, the mutation
in the lid severely lowers the activation of the peptidase activity of
the proteasome, suggesting that the disorganization caused in the RP by
the depletion of Rpn6p subunit also hinders its ability to properly
open the CP channel.
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ACKNOWLEDGEMENTS |
---|
We thank M. C. Fernández for expert technical assistance. We thank E. Bailly, A. Bueno, J. H. Hegemann, D. Koshland, C. Mann, E. Schowb, and A. Toh-e for gifts of strains, antibodies, or protocols. We gratefully thank Marion Schmidt for helpful discussions and critical reading of the manuscript and the 310 (Centro de Biología Molecular) and Finley (Harvard Medical School) laboratory members for stimulating discussion and advice.
![]() |
FOOTNOTES |
---|
* This work was supported by Grant PM1999-0108 from the Ministerio de Ciencia y Tecnología (Spain) and National Institutes of Health Grant GM43601 (to D. F.) and by an institutional grant to the Centro de Biología Molecular "Severo Ochoa" from Fundación Ramón Areces (Madrid).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 Formación Personal Investigador
fellowship from the Comunidad Autónoma de Madrid.
¶ To whom correspondence should be addressed. Tel.: 34-91-397-8676; Fax: 34-91-397-4799; E-mail: miguel.remacha@uam.es.
Published, JBC Papers in Press, December 16, 2002, DOI 10.1074/jbc.M209420200
2 P. G. Santamaría, J. P. G. Ballesta, and Miguel Remacha, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: Ub, ubiquitin; CP, core particle; RP, regulatory particle; CSN, COP9-signalosome complex; HA, hemagglutinin; DAPI, 4',6-diamindine-2'-phenylindole; MOPS, 4-morpholinepropanesulfonic acid; APC/C, anaphase-promoting complex/cyclosome.
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