From the Department of Microbiology and Immunology, University of California, San Francisco, California 94143-0414
Received for publication, November 20, 2002, and in revised form, January 30, 2003
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ABSTRACT |
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The polyamine biosynthetic enzyme ornithine
decarboxylase (ODC) is degraded by the 26 S proteasome via a
ubiquitin-independent pathway in mammalian cells. Its degradation is
greatly accelerated by association with the polyamine-induced
regulatory protein antizyme 1 (AZ1). Mouse ODC (mODC) that is expressed
in the yeast Saccharomyces cerevisiae is also rapidly
degraded by the proteasome of that organism. We have now carried out
in vivo and in vitro studies to determine
whether S. cerevisiae proteasomes recognize mODC degradation signals. Mutations of mODC that stabilized the protein in
animal cells also did so in the fungus. Moreover, the mODC degradation
signal was able to destabilize a GFP or Ura3 reporter in GFP-mODC and
Ura3-mODC fusion proteins. Co-expression of AZ1 accelerated mODC
degradation 2-3-fold in yeast cells. The degradation of both mODC and
the endogenous yeast ODC (yODC) was unaffected in S. cerevisiae mutants with various defects in ubiquitin metabolism, and ubiquitinylated forms of mODC were not detected in yeast cells. In
addition, recombinant mODC was degraded in an ATP-dependent manner by affinity-purified yeast 26 S proteasomes in the absence of
ubiquitin. Degradation by purified yeast proteasomes was sensitive to
mutations that stabilized mODC in vivo, but was not
accelerated by recombinant AZ1. These studies demonstrate that cell
constituents required for mODC degradation are conserved between
animals and fungi, and that both mammalian and fungal ODC are subject
to proteasome-mediated proteolysis by ubiquitin-independent mechanisms.
The 26 S proteasome is the major neutral protease of the
cytoplasmic and nuclear compartments of eukaryotic cells. It is a multisubunit, ATP-dependent, protease composed of at least
two subcomplexes: a barrel-shaped 20 S core particle that sequesters the proteolytic active sites from the intracellular environment, and a
19 S regulatory particle that contains activities required for
recognition, unfolding, and translocation of substrates to the interior
of the core particle (1). Its substrate proteins include major
regulators of cell growth and differentiation, and also proteins that
fail to fold into a native conformation. The proteasome needs therefore
to recognize appropriate substrates accurately to support proper timing
of cellular events and to eliminate proteins that have failed to follow
normal folding pathways. Many of these short-lived proteins are
targeted to the proteasome by the conjugation of ubiquitin, an
evolutionarily conserved 76-amino acid globular protein (2). The
efficient recognition of ubiquitinylated substrates by the proteasome
requires, minimally, the formation of a tetraubiquitin chain on the
substrate protein (3). The polyubiquitin signal is in turn recognized
by a receptor site that includes the Rpt5/S6' ATPase subunit of the
regulatory particle (4). Additionally, the proteasome has the capacity
to recognize a class of substrates that do not require ubiquitin
modification for their regulated degradation (5). These include the
cyclin-dependent kinase inhibitor p21 and ornithine
decarboxylase (ODC),1 which
catalyzes the initial step in polyamine biosynthesis.
In most eukaryotic cells, ODC is subject to regulation by spermidine
and spermine, the end products of the polyamine biosynthetic pathway.
These act not through allosteric feedback regulation, the usual
mechanism, but by changing the abundance of the ODC protein (6). This
form of feedback regulation is accomplished by an autoregulatory
circuit composed of ODC, polyamines, and the regulatory protein
antizyme 1 (AZ1) (7). Excess polyamines induce a +1 translational
frameshift of the AZ1 mRNA that is required to align a short
upstream open reading frame (ORF) with a downstream ORF encoding the
functional protein (8). AZ1 binds the ODC monomer, dissociating the
enzymatically active ODC homodimer and thereby inhibiting its activity
(9). AZ1 binding exposes a COOH-terminal degradation signal in the ODC
protein that leads to an increased rate of degradation of ODC by the
proteasome (10). The AZ1-dependent degradation of ODC by
the proteasome is remarkable in that it occurs in mammalian cells
independently of the ubiquitinylation of ODC. Both in vivo
data (11, 12) and in vitro data (13, 14) support this conclusion.
The ODC-AZ regulatory circuit has been best characterized in mammalian
cells, but functional AZ homologs have also been described in a number
of other organisms, including the nematode Caenorhabditis elegans (15), the fly Drosophila melanogaster (16),
several filamentous fungi, and the fission yeast
Schizosaccharomyces pombe (15, 17). However, no AZ homolog
is evident in the genome sequence of the budding yeast
Saccharomyces cerevisiae (18). In this organism, polyamine
regulation of ODC resembles that of mammalian cells in that
excess polyamines increase the rate of ODC degradation (19). Polyamine
regulation in S. cerevisiae also involves de novo
protein synthesis (20), a requirement of an AZ-like regulatory
mechanism, but not of polyubiquitinylation. Both yeast and mouse ODC
are rapidly degraded by the 26 S proteasome in S. cerevisiae
(19, 21).
Proteasome structure is highly conserved between mammals and fungi, and
so too is the general use of ubiquitin modification as a marker for
recognition of substrates. We ask here whether, and to what extent,
mammalian and yeast proteasomes also share the capacity to recognize
and respond to the structural hallmarks that are used in mammalian
cells for the constitutive and regulated degradation of ODC. We show
here that for both yeast and mouse ODC, degradation in yeast cells does
not depend on ubiquitin. We found that recognition of mouse ODC
requires similar structural and functional elements, regardless of the
source of the proteasome, implying that ubiquitin-independent
substrates, like those that depend on ubiquitin, must utilize a
conserved discriminatory capacity of the proteasome.
Yeast Strains--
Yeast strains were maintained and manipulated
using standard protocols (22). For analysis of FLAGmODC and
yODCFLAG turnover (reported in Figs. 1, 3, and 4), the
appropriate expression vectors were transformed into strain Y13
(MAT Plasmid Construction--
All plasmid constructions utilized
standard molecular biology techniques (29), and the identities of DNA
fragments generated by PCR were verified by sequencing. The sequences
of primers used for PCR constructions are available upon request. FLAG
epitope tags were added to the termini of mODC (30),
mODCC441A (31), and yODC (32) by PCR using primers that
also appended flanking restriction sites. The PCR products were
digested with the appropriate restriction endonucleases and ligated
into the similarly digested expression vectors of Mumberg et
al. (33). These vectors include promoters of various strengths for
heterologous expression in S. cerevisiae. We designed a
similar vector containing the yODC (SPE1) promoter by PCR
amplification of a region containing the 574 nucleotides upstream of
the yODC ORF in the SPE1 gene while appending 5'
SacI and 3' XbaI sites. The ADH
promoter was removed from the p414ADH vector (33) by digestion with
SacI and XbaI, and replaced with the similarly
digested PCR product containing the SPE1 promoter by
ligation. The resulting TRP1-marked CEN/ARS
vector was designated p414SPE1. The COOH-terminal FLAG-tagged yODC ORF
was cloned as a BamHI-XhoI PCR product into
p414SPE1 to create p414SPE1-yODCF. To generate COOH-terminal
truncations of mODC, the mODC ORF was amplified using a common sense
primer complementary to the NH2 terminus of mODC (including
the FLAG epitope tag and flanking restriction sites) and antisense
primers that introduce stop codons at Phe425
(mODC
GFP-mODC fusions were constructed by splice overlap extension PCR
(SOE-PCR) using a pGFPuv vector (BD Sciences
Clontech) as a template. A PCR fragment containing
the COOH-terminal half of the GFP ORF was generated by a sense primer
overlapping the XhoI site within the GFPuv coding region,
and an antisense primer complementary to the COOH terminus of GFPuv and
amino acids 425-461 of mODC. A second PCR fragment was generated by
amplification of the COOH terminus of the mODC or mODCC441A
coding region using a sense primer complementary to the antisense primer used to generate the GFP fragment and an antisense primer that
appended an EcoRI site following the stop codon of the mODC ORF. Following SOE-PCR, the resulting DNA fragments were digested with
XhoI and EcoRI, and ligated into a similarly
digested pGFPuv vector from which the fragment containing the COOH
terminus of GFPuv had been removed. The ORFs encoding GFPuv and
GFP-mODC fusion proteins were subcloned from the pGFPuv-based vectors
into yeast expression vectors as XbaI-EcoRI fragments.
Ura3-mODC fusions were constructed similarly to the GFP-mODC fusions by
SOE-PCR. The coding region of URA3 was amplified from the
pRS306 vector (34) using a sense primer that appended an NH2-terminal FLAG epitope tag and a flanking
BamHI site, and an antisense primer complementary to the
COOH terminus of Ura3 and amino acids 425-461 of mODC. A DNA fragment
containing the COOH terminus of mODC was generated using a sense primer
complementary to the antisense primer used to amplify the
URA3 coding region and an antisense primer that appended an
EcoRI site following the stop codon of the mODC ORF.
Following SOE-PCR, the DNA fragment encoding Ura3-mODC was digested
with BamHI and EcoRI and ligated into similarly
digested yeast expression vectors.
Metabolic Labeling, Immunoprecipitations, and
Immunoblotting--
Pulse-chase analysis was carried out similarly to
the protocol described by Suzuki and Varshavsky (35). 10-ml cultures of yeast transformants were grown to midexponential growth phase (A600 0.5-1). Cells were harvested by
centrifugation (3 min, 2000 × g), and washed twice in
1 ml of SD medium lacking methionine (SD
For immunoprecipitations of FLAG-tagged proteins, normalized volumes of
lysates were incubated for 2 h at 4 °C with 20 µl of a 50%
slurry of anti-FLAG M2 affinity gel (Sigma) in lysis buffer.
Immunoprecipitations of Leu-
For immunoblot analysis 5-ml cultures of yeast cells were grown to
midexponential growth phase in SD medium lacking the appropriate nutritional supplements. Cells were collected by centrifugation (3 min,
2000 × g), and washed in 5 ml of water, followed by
resuspension in 200 µl of phosphate-buffered saline + 1% Triton
X-100. The cell suspension was added to 200 µl of glass beads and
lysed by 3 pulses of 20 s duration in a Mini-beadbeater with
intermittent cooling on ice. Cell debris was removed by centrifugation
(13,000 × g for 5 min at 4 °C), and volumes of
lysates containing 20 µg of total protein were fractionated by
SDS-10% PAGE. Fractioned proteins were transferred to nitrocellulose
filters, and developed using the Amersham ECL detection kit and
protocol. Filters were blocked in Tris-buffered saline + 1% Triton
X-100 (TBS-T) including 5% powdered low-fat milk and 5% bovine serum
albumin, and washed 3 times for 5 min with TBS-T. FLAG-tagged proteins
were detected with mouse anti-FLAG M2 antibody (Sigma, 1:2000 dilution)
and sheep anti-mouse Ig-horseradish peroxidase conjugates
(Amersham Biosciences, 1:15,000 dilution).
For detection of ubiquitin conjugates, cells were grown and harvested
as described above, but lysis and immunoprecipitations were carried out
in buffers containing 50 mM N-ethylmaleimide. Hemagglutinin-ubiquitin conjugates were detected, following
fractionation by SDS-PAGE and transfer to nitrocellulose, by incubation
with anti-HA-horseradish peroxidase conjugates (Santa Cruz
Biotechnology, 1:1000 dilution).
Proteasome Purification--
Proteasomes were affinity purified
from RJD1144 cells as described (28). Normally, this purification
protocol involves separating tagged proteasome complexes from extracts
using anti-FLAG antibody conjugated to agarose beads followed by
elution of the complexes with FLAG peptide. We have found that the FLAG
peptide causes activation of the normally gated 20 S core particle that
results in a reproducible ATP-independent degradation of
mODC.2 To avoid exposure to
the FLAG peptide, we used proteasome complexes still associated with
the antibody-agarose, a form active for ATP-dependent
proteolysis of mODC. Briefly, cells were grown to early stationary
phase (A600 2-2.5) in 2 liters of SD medium and harvested by centrifugation (2000 × g, 20 min). The
cells were pooled and washed in 100 ml of ice-cold water. The cell
pellet was resuspended in an equal volume of buffer A (50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM
MgCl2, 10% glycerol) containing 5 mM ATP and
drop frozen in liquid N2 in a mortar resting in a bed of
dry ice. The cells were ground with a pestle for 30 min with periodic additions of liquid N2 to keep the material frozen. The
ground cells were thawed and most cell debris was removed from the
lysate by centrifugation (4,000 × g, 10 min), followed
by a second centrifugation (30,000 × g, 20 min) to
remove remaining debris. 10 ml of the cleared lysate (~200 µg of
protein) was incubated with 300 µl of anti-FLAG M2 affinity gel for
90 min at 4 °C on a rocking platform. The agarose beads were
collected by centrifugation (2000 × g, 10 min),
transferred to a microcentrifuge tube, and washed with 10 ml of buffer
A containing 2 mM ATP and 0.2% Triton X-100, followed by 2 washes with 1 ml of buffer A with 2 mM ATP. The
proteasome-agarose beads were resuspended in an equal volume of buffer
A with 2 mM ATP and used for degradation assays. Total
protein bound to the anti-FLAG agarose was estimated by elution in 1 M NaCl followed by protein determination of the resulting eluate.
In Vitro Degradation Assays--
The degradation of mODC by
purified proteasomes was followed by a released radiolabel assay.
Recombinant radiolabeled mODC was produced in Escherichia
coli as described
elsewhere.3 Degradation
assays were performed in 20-µl reaction volumes at 37 °C and
contained 50 mM Tris-HCl, pH 7.5, 5 mM
MgCl2, 1 mM ATP, 10 mM KCl, 10%
glycerol, an ATP regenerating system (2 mM dithiothreitol, 10 mM creatine phosphate, 1.6 mg/ml creatine kinase), 2 mg/ml bovine serum albumin and proteasomes. Reactions were initiated by
addition of substrate and quenched by addition of 140 µl of 20%
(w/v) trichloroacetic acid. The trichloroacetic acid-insoluble material
was removed by centrifugation (14,000 × g, 30 min) at 4 °C, and 150 µl of the supernatant was removed and released
counts were measured in a liquid scintillation counter. Percentage
degradation of radiolabeled mODC was determined as the released
counts/min minus background counts/min divided by total input
counts/min. Total input counts were determined by substituting water
for trichloroacetic acid in the reactions. Background counts/min were
determined in reactions that excluded proteasomes, and were typically
~1% of total input counts.
Fluorescence Measurements--
The fluorescence of GFPuv and
GFP-mODC proteins in yeast transformants was measured in arbitrary
units with a TD-700 laboratory fluorometer (Turner Designs) using a
394-nm excitation filter and a 510-nm emission filter. Fluorescence
measurements were made in cultures at exponential growth phase
(A600 0.5-1), and background autofluorescence
was measured in an empty vector transformant and subtracted from raw
fluorescence measurements.
Carboxyl-terminal Recognition Elements of Mouse ODC Are Recognized
in Yeast--
To facilitate studies of mODC degradation in S. cerevisiae, we appended a single copy of the FLAG epitope tag to
the NH2 terminus of mouse ODC (FLAGmODC). The
tagged mODC was expressed from yeast centromeric expression vectors
(33) in a spe1
Mouse ODC expressed in yeast cells is rapidly degraded by the 26 S
proteasome (21). We compared, in yeast transformants, the degradation
of FLAG-tagged wild type mODC expressed from either the yeast
ADH promoter or the stronger GPD promoter. We
found that wild type FLAGmODC expressed from the
ADH promoter was easily detectable by immunoblotting (Fig.
1A) and rapidly degraded in
spe1
We used vectors containing the ADH promoter to compare
turnover of wild type and mutant forms of mODC in yeast cells. The mODC
mutants analyzed included truncated forms lacking the COOH-terminal 5 or 37 amino acids of the mODC, and full-length mODC containing a
Cys441 to Ala mutation. The COOH-terminal 37 amino acids of
mODC (residues 425-461) contains a signal required for its rapid
degradation in animal cells or in vitro (10, 38), and both
Cys441 and the 5 terminal residues in this region are
critical for its function (31, 39). Truncation of 5 or 37 residues from
the COOH terminus, or the mutation of Cys441 to Ala, led to
greater expression of these proteins than of the wild type
FLAGmODC in exponentially growing cultures (Fig.
1A), and significant stabilization of these proteins
throughout a 1-h chase period following metabolic labeling (Fig.
1B). Thus, COOH-terminal mutations that alter mODC
degradation in animal cells similarly affect this process in yeast cells.
The Carboxyl Terminus of Mouse ODC Is a Portable Proteasome
Recognition Element in Yeast--
Because mODC is unstable in yeast
cells, and truncation of its COOH terminus leads to stabilization, we
asked whether this region of mODC conferred rapid turnover when
attached to either a heterologous or endogenous protein expressed in
S. cerevisiae. The fusion of the COOH-terminal 37 amino
acids of mODC to the COOH terminus of Trypanosoma brucei ODC
or GFP leads to the rapid turnover of these otherwise stable proteins
in mammalian cells (40, 41). We attached the COOH-terminal 37 amino
acids of mODC (425-461) to the COOH terminus of either GFP from
Aequorea victoria, or orotidine-5'-monophosphate
decarboxylase encoded by the URA3 gene of S. cerevisiae. We constitutively expressed the GFP-mODC fusion
protein and unmodified GFP in yeast transformants from the
ADH1 promoter. Immunoblot analysis with an anti-GFP antibody (Fig. 2A) showed that the
steady-state level of GFP-mODC was greatly reduced compared with that
of unmodified GFP in the yeast transformants. GFP fluorescence was
similarly reduced in cells expressing the GFP-mODC fusion (Fig.
2A).
We measured the degradation of GFP and the GFP-mODC fusion in yeast
cells directly by pulse-chase analysis. Whereas unmodified GFP was
stable throughout the 30-min chase period, the GFP-mODC fusion was
degraded with a half-life of ~10 min (Fig. 2B), confirming that the COOH-terminal 37 amino acids of mODC behaved similarly as a
degradation signal in fungal and mammalian cells. We conclude that the
decreased expression of GFP, to which the COOH-terminal portion of mODC
has been appended, was caused by its increased rate of degradation. We
found similar effects when we attached these mODC sequences to the COOH
terminus of the S. cerevisiae URA3 ORF, which
encodes a normally stable protein (Fig. 2C). Thus, the mODC
COOH terminus appears capable of acting in cis to impart instability to a number of unrelated proteins in yeast cells.
Whereas appending the COOH terminus of mODC could import a
cis-acting degradation signal to GFP (or Ura3) functionally
akin to that present in native mODC, it is possible that the attachment of these sequences could result in some other effect, such as misfolding, which could result in accelerated degradation. To rule this
out, we introduced a C441A mutation in the GFP- and Ura3-mODC fusion
proteins. Pulse-chase analysis demonstrated that the C441A mutation led
to the stabilization of both fusion proteins (Fig. 2, B and
C). This mutation should prevent degradation effects specific to the mODC COOH terminus, but not more general effects, such
as perturbation of structural integrity, which could lead to rapid
degradation of the fusion protein. Additionally, the GFP-mODC fusion
protein was fluorescent (Fig. 2A), and the Ura3-mODC fusion
protein supported the growth of a ura3 mutant (data not shown), indicating that the mODC extensions did not perturb the normal
functions of these proteins. We therefore conclude that the
destabilization of the fusion proteins is because of the presence of
the mODC COOH terminus specifically, rather than misfolding or other
artifactual characteristics of the fusion protein.
To confirm that the reduction in steady-state levels of the fusion
proteins was because of increased degradation by the 26 S proteasome,
we examined expression of the GFP-mODC fusion protein in a pre1-1
pre2-2 mutant. This mutant carries mutations in the Effects of Polyamine Pool Repletion and Antizyme on Turnover of
Mouse and Yeast ODC--
In mammalian cells, increases in cellular
polyamine pools leads to the rapid degradation of mODC, a process
mediated by de novo synthesis of the regulatory protein AZ1
in response to polyamines. In S. cerevisiae a similar
mechanism apparently functions in the regulation of endogenous yODC
(20), although no AZ1 homolog has yet been identified in this organism
(18). We asked whether this S. cerevisiae
polyamine-responsive regulatory factor acted on mODC that was expressed
in yeast cells, and if heterologous expression of a mammalian AZ1
further accelerated mODC degradation in these cells. We compared the
effect of polyamine administration on the expression of
FLAGmODC and COOH-terminal tagged yODCFLAG,
also expressed from a centromeric vector in the
spe1
We next determined the effect of expression of AZ1 on the degradation
of FLAGmODC in yeast cells. For these studies, we used a
rat AZ1 cDNA in which the two partial ORFs comprising full-length
AZ1 had been aligned by a single base deletion (8). This AZ1 ORF
(AZ1
To verify that the decreased levels of FLAGmODC in
transformants in which AZ1 was also expressed were because of
accelerated degradation, we measured the effects of AZ1 co-expression
on FLAGmODC turnover by pulse-chase analysis. The results
showed that the half-life of FLAGmODC decreased from ~10
min in a transformant carrying an empty vector control (Fig.
4C, top panel) to ~4 min in transformants co-expressing FLAGAZ1 (Fig. 4, C, bottom
panel, and D). We conclude that AZ1 expression causes a
modest, but reproducible acceleration of mODC turnover in yeast cells.
In contrast with the effect of AZ1 on mODC in mammalian cells, in which
a small amount of AZ1 relative to mODC produces a large effect on
turnover, an excess of AZ1 leads to a modest increase in turnover of
mODC in S. cerevisiae.
Turnover of Mouse and Yeast ODC Is Independent of
Ubiquitin--
In both cultured mammalian cells (12), and in
vitro (14), AZ1-dependent degradation of mODC by the
proteasome is independent of the ubiquitin system. To determine whether
ubiquitinylated forms of FLAGmODC were present in yeast
cells, FLAGmODC was immunoprecipitated from yeast
transformants expressing either ubiquitin (Ub) or a hemagglutinin
epitope-tagged form of ubiquitin (HAUb). As a positive
control, transformants expressing Leu-
Ubiquitin conjugates to a particular protein may be difficult to detect
because of their low abundance, transience, or the action of
deubiquitinylating enzymes before or after cell lysis. To verify the
inference that ubiquitin was not involved in proteasomal degradation of
FLAGmODC, we examined the degradation of this protein in
S. cerevisiae mutants affecting ubiquitin metabolism. The
uba1-2 mutant carries an insertion of a mini-transposon 84 bp upstream of the translation start site of the UBA1 gene
(26). The UBA1 gene encodes the ubiquitin-activating enzyme,
E1, and is essential for viability of S. cerevisiae (46).
The uba1-2 allele reduces wild type E1 function, and causes
defects in a variety of ubiquitin-dependent processes (26).
The DOA4 gene encodes a deubiquitinylating enzyme that
interacts with the 26 S proteasome and acts late in the proteolytic pathway (27, 47). Doa4 is involved in the recycling of ubiquitin from
proteolytic substrates, and in doa4
Using the criteria described above for FLAGmODC, we asked
whether ubiquitin plays a role in the degradation of
yODCFLAG in response to excess polyamines. We measured the
half-life of yODCFLAG in uba1-2 and
doa4 In Vitro Degradation by Yeast Proteasomes Reproduces Salient
Characteristics of in Vivo Degradation--
Both the
AZ1-dependent (14) and AZ1-independent3
degradation of mODC can be reconstituted in vitro with 26 S
proteasomes from mammalian sources. We used affinity-purified yeast
proteasomes to determine whether ubiquitin-independent mODC degradation
could be replicated in a yeast in vitro system. Yeast
proteasomes were isolated from a strain carrying a COOH-terminal
FLAG-His6 tagged form of the Pre1
Using the immunoaffinity purified yeast proteasomes and recombinant
mODC produced in E. coli as substrate, we verified that degradation of mODC was linear with time and increasing proteasome concentrations, and inhibited >90% by epoxomicin (Supplemental Fig.
1). Epoxomicin is a specific inhibitor of the proteasome that
covalently binds to its catalytic sites in the 20 S core particle (50).
As expected for a substrate of the 26 S proteasome, mODC degradation
was ATP-dependent, and required association of the 19 S
regulatory particle with the 20 S core (Fig. 7B). We compared the effects of mODC mutations that limit its degradation in
cells on degradation in vitro using either yeast or rat
proteasomes (Fig. 7C). Mutations that truncated the last 5 amino acids of the mODC COOH terminus or altered Cys441 had
similar inhibitory effects on degradation by proteasomes from either source.
Surprisingly, the addition of AZ1, even in large stoichiometric excess,
had no stimulatory effect on mODC degradation by yeast 26 S proteasomes
(Fig. 7D). Under similar assay conditions, AZ1 stimulated
the degradation of mODC by rat liver proteasomes ~6.5-fold (Fig.
7D). Both yeast and rat proteasomes showed similar
degradative activity toward mODC in the absence of AZ1, but AZ1
significantly stimulated only degradation by the mammalian protease. We
conclude that mODC degradation by the yeast 26 S proteasome can be
reconstituted in vitro, in the absence of the
ubiquitinylation of mODC. However, degradation in the purified system
differed from both in vivo degradation in yeast cells and
in vitro degradation using mammalian-derived proteasomes in
that AZ1 had no stimulatory effect on mODC degradation.
For the majority of labile proteins, post-translational
modification with polyubiquitin constitutes the necessary and
sufficient marker for proteasomal recognition. The addition of
polyubiquitin chains depends on a complex series of enzymes (2). The
task of substrate identification and marking is thus removed from the proteasome itself and devolves instead on a series of ubiquitin activators and transferases. Such delegation of executive authority seems to limit the proteasome to a straightforward binary decision: if
and only if a protein bears a polyubiquitin marker of the requisite size, it is to be recognized, unfolded, inserted into a hollow catalytic chamber, and there hydrolyzed to peptides.
This view of proteasome function likely understates its discriminatory
capacity. Both prokaryotes and eukaryotes contain compartmentalized proteases that accomplish substrate recognition by association with
regulatory complexes. The prokaryotic E. coli Clp/Hsl
proteolytic complexes are composed of two stacked hexameric rings of a
protease subunit flanked on one or both sides by hexamers of a
regulatory ATPase subunit. These ATPases each recognize distinct and
limited sets of substrate proteins (51). The greater structural
complexity of the eukaryotic proteasome 19 S regulator suggests that it
is capable of more elaborate forms of substrate recognition and
processing. The 19 S complex includes two subassemblies. The base is
juxtaposed to the ends of the 20 S core complex and contains six ATPase
proteins plus two additional proteins. The lid, hinged to the base and positioned distally, contains 12-14 other proteins. The mammalian proteasome 19 S regulatory complex, in addition to making a binary decision based on the presence or absence of a ubiquitin chain, has the
capacity to edit polyubiquitin chains, perhaps performing thereby a
proofreading function (52). The proteasome also has direct interactions
with the enzymes that carry out late steps in the process of adding
ubiquitin chains (28, 53). Additionally, the proteasome has the
capacity to recognize a class of substrates that do not require
ubiquitin modification for their regulated degradation. This class of
substrates includes the cyclin-dependent kinase inhibitor
p21 (54) and ODC (14). We have here tested the capacity of yeast
proteasomes to recognize phylogenetically distant ubiquitin-independent
degradation signals, those associated with mODC.
Mamroud-Kidron and Kahana (21) showed that mODC was rapidly degraded in
yeast and provided genetic evidence that the 26 S proteasome was the
protease responsible. We show now that mODC degradation has similar
structural requirements in yeast and animal cells, namely its
COOH-terminal five amino acids and Cys441, that these
elements can be exported to an otherwise stable protein, that
degradation occurs without prior ubiquitinylation in yeast as well as
animal cells, and that purified yeast proteasomes have similar
capabilities. The present studies therefore demonstrate both in
vivo and in vitro that S. cerevisiae
proteasomes conserve salient characteristics of mODC recognition.
Our present studies contribute to answering a further question. Can
degradation of mODC take place without AZ1? In vivo evidence has been difficult to gather because it is hard to fully exclude the
action of residual AZ1 in animal cells. Using yeast as a foreign host
resolves this issue. There is no obvious AZ1 homolog in S. cerevisiae, and the unidentified yeast ODC regulatory protein does
not act on mODC when induced by polyamine addition, yet mODC is rapidly
degraded in these cells. This degradation shows the same structural
requirements as in animal cells. Thus, in a cell type lacking any
obvious AZ1 or other polyamine-responsive regulatory activity that can
act on mODC, this protein is subject to rapid degradation by the proteasome.
In vitro evidence for AZ-independent degradation of mODC has
also been difficult to obtain, likely because of the use of assay methods insufficiently sensitive to accurately measure limited substrate degradation. We have now demonstrated in a purified system
that the degradation of mODC by yeast or rat proteasomes occurs
in vitro in the absence of AZ1. Given the relatively low levels of AZ1-independent mODC degradation using our in
vitro assays (<10%), only the combined use of a released
radiolabel assay, a substrate of high specific radioactivity, and
concentrated proteasomes allow its detection. Because the occurrence of
AZ-independent degradation of ODC has previously been hard to
establish, the question of whether it requires ubiquitin has also been
difficult to answer. This question is now clearly resolved: regardless
of whether AZ1 is or is not present, degradation of ODC takes place without the participation of ubiquitin.
Although AZ1 is dispensable for mODC degradation in yeast cells, AZ1
expression does produce an increase in the rate of mODC degradation.
This 2-3-fold stimulation of mODC degradation is more modest than the
~10-fold effect seen in animal cells (55). However, the 10-min
half-life of mODC in yeast is also more rapid than in the ~60-min
half-life of this protein in animal cells in the absence of AZ1
induction. Perhaps the more rapid basal degradation of mODC in yeast
cells mutes its response to AZ1. Strikingly, the AZ1 stimulation of
mODC turnover is lost in our purified in vitro system, but
retained in purified systems utilizing proteasomes from mammalian
sources (14).
The structural features of mammalian ODC that are required for its
degradation are not present in the otherwise highly conserved yeast
homolog. When the two proteins are aligned, mODC is seen to contain a
COOH-terminal extension, not present in yODC. Conversely, yODC contains
an NH2-terminal extension not found in the mammalian enzyme. The mODC degradation signal resides in its COOH terminus (38,
40), and the NH2 terminus of yODC is required for its rapid
degradation (42). Although the mODC degradation signal is recognized by
both mammalian and yeast proteasomes, yODC is stable in mammalian cells
(42), suggesting either that its degradation signal is specific for
yeast proteasomes, or that interaction with the yeast polyamine
regulator is indispensable for yODC degradation.
Even though no AZ homolog has yet been identified in S. cerevisiae, a polyamine-ODC-AZ regulatory circuit may yet exist in this organism. Several observations strengthen this conjecture. Polyamines increase the turnover of ODC in yeast cells (19), this
regulation requires new protein synthesis (20), and, as shown here, the
regulated turnover of yODC is ubiquitin-independent. It has also been
recently reported that the basal turnover rate of yODC (in the absence
of excess polyamines) is not impaired in mutants that alter ubiquitin
metabolism (42). The only other substrate of the proteasome shown to be
degraded in S. cerevisiae independently of ubquitinylation
is Rpn4. Rpn4 is a transcriptional activator of genes encoding subunits
of the proteasome as well as other genes (56, 57). The Rpn4 protein is
short-lived and interacts with the Rpn2 subunit of the base of the 19 S
regulatory particle (58).
The finding that yeast provides an appropriate milieu for studies of
mammalian ODC degradation by the 26 S proteasome will facilitate future
studies. For example, the apparent discrepancy between the capacity of
AZ1 to accelerate degradation in vivo but not in
vitro suggests the participation of additional components of the
degradative system that are excluded upon purification. Utilizing the
genetic methods available in yeast should help to reveal the identity
and roles of ancillary factors that influence degradation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
his3 leu2 trp1 ura3
spe1
::HisG
spe2
::LEU2), a polyamine auxotroph
(19). Growth in defined medium of polyamine auxotrophic strains was
maintained by addition of 0.1 µM spermidine, a
concentration insufficient for repression of endogenous yODC; the level
of spermidine in the medium was increased to 1 mM to induce
repression of yODC. Vectors for the expression of GFP or Ura3 fusion
proteins (Fig. 2) were transformed into strain MHY501
(MAT
his3-
200 leu2-3,112 lys2-801
trp1-1 ura3-52) (23) or the proteasome mutant WCG4-11/22a (MATa his3-11,15 leu2-3,112 ura3 pre1-1 pre2-2) and its
congenic wild type strain WCG4a (MATa his3-11,15 leu2-3,112
ura3) (24). An rpn4
mutant, MHY74 (MAT
his3-
200 leu2-3,112 lys2-801 trp1-1 ura3-52
rpn4
::kanMX4) was created by replacement
of the entire RPN4 ORF of strain MHY501 with a
kanMX4 cassette from pRS400 generated by PCR as described
(25). To examine the effects of ubiquitin metabolism on ODC turnover
(Fig. 6), FLAGmODC, yODCFLAG, or
Leu-
-galactosidase vectors were transformed into MHY1409 (MAT
his3-
200 leu2-3,112 lys2-801
trp1-1 ura3-52 gal2 uba1-2 (mTn URA3)) (26), MHY623
(MAT
his3-
200 leu2-3,112 lys2-801 trp1-1 ura3-52 doa4-
1::LEU2)
(27), or MHY501. Proteasomes were affinity-purified from strain RJD1144
(MATa his3-
200 leu2-3,112 lys2-801
trp1
63 ura3-52
PRE1FH::YIplac211 (URA3))
(28).
425-461) or Ala457 (mODC
457-461). The AZ1
T
ORF was cloned from pGEM4Z/
T (8) as a
HindIII-EcoRI fragment into similarly digested
URA3-marked Mumberg vectors.
Met). The cells were
resuspended in 0.4 ml of SD
Met and labeled for 5 min at 30 °C
with 200 µCi of a L-[35S]methionine and
L-cysteine mixture (Expre35S35S
protein labeling mixture, PerkinElmer Life Sciences). To terminate incorporation of radiolabel and initiate a chase, cells were collected by brief centrifugation in a microcentrifuge, and resuspended in 0.4 ml
of SD medium containing 10 mM methionine, 1 mM
cysteine, and 0.5 mg/ml cycloheximide. Incubation was continued at
30 °C. At each time point 100 µl of cells were removed and
transferred to a 2-ml screw-top microcentrifuge tube containing 700 µl of ice-cold lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride) and 500 µl of 0.5-mm
glass beads. Cells were lysed by 4 pulses of 30 s duration in a
Mini-beadbeater (Biospec Products), with cooling on ice between pulses.
Lysates were cleared by centrifugation (13,000 × g, 10 min at 4 °C), and the amount of trichloroacetic acid-insoluble
radioactivity was determined. The volumes of lysates were adjusted to
contain equivalent amounts of trichloroacetic acid-insoluble
radioactivity for immunoprecipitations.
-galactosidase or GFP were carried out
by incubation of lysates with 1 µl of anti-
-galactosidase antibody
(Promega) or anti-GFP antibody (BD Sciences
Clontech) for 2 h at 4 °C, followed by the
addition of 20 µl of a 50% slurry of protein G-Sepharose (Amersham
Biosciences) and a further 1-h incubation at 4 °C.
Immunoprecipitates were collected by brief centrifugation (2000 × g, 20 s), and washed 4 times with 1 ml of lysis buffer
containing 0.1% SDS. The immunoprecipitates were resuspended in SDS
sample buffer (29), heated to 100 °C for 5 min, and fractionated by
SDS-PAGE on 10% (FLAGmODC, yODCFLAG,
FLAGUra3 fusions, or GFP fusions) or 7.5%
(
-galactosidase) polyacrylamide gels. Radiolabeled proteins were
visualized by autoradiography, and quantified using Scion Image
software (Scion Corp.).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
spe2
mutant. This mutant
lacks enzymes that catalyze key steps in polyamine biosynthesis in
S. cerevisiae, and cannot accumulate spermidine, the
effector of polyamine regulation in this organism. Polyamine pools are
thus accessible to experimental control in this genetic background.
spe2
cells with a half-life of ~10
min, as measured by pulse-chase analysis (Fig. 1B). The
expression of FLAGmODC protein from a vector containing the
more active GPD promoter led to a 3-fold stabilization of
FLAGmODC in those transformants (half-life = ~30
min, Fig. 1B), suggesting that overexpression interferes
with normal degradation of mODC as it does with turnover of endogenous
yODC (19, 36, 37).
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Fig. 1.
COOH-terminal mODC mutants are stable in
yeast cells. A, immunoblot analysis with anti-FLAG
antibody (Sigma) of spe1 spe2
cells
transformed with p414ADH vectors expressing wild type
FLAGmODC or COOH-terminal mutants. B,
pulse-chase analysis of FLAGmODC in
spe1
spe2
transformants. Wild type
FLAGmODC was expressed from the ADH
(PADH) or GPD (PGPD)
promoters in p414 vectors. COOH-terminal mutants were all expressed
from the ADH promoter of p414ADH. Epitope-tagged mODCs were
immunoprecipitated with anti-FLAG antibody.
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Fig. 2.
GFP- and Ura3-mODC fusion proteins are
unstable in yeast cells. A, immunoblot analysis with
anti-GFP antibody of MHY501 transformed with p416ADH vectors expressing
wild type GFPuv or a GFPuv fusion to amino acids 425-461 of mODC.
The GFP fluorescence of transformants expressing the corresponding
proteins is indicated in arbitrary fluorescence units. B,
pulse-chase analysis of GFPuv and GFP-mODC fusion protein turnover in
yeast transformants. GFPuv and GFP-mODC fusions were immunoprecipitated
with anti-GFP antibody and protein G-Sepharose. Arrows
indicate the position of the appropriate proteins. C,
pulse-chase analysis of FLAGUra3-mODC and
FLAGUra3-mODCC441A fusion protein turnover in
yeast transformants. FLAGUra3-mODC fusions were
immunoprecipitated with anti-FLAG M2 affinity gel. D,
immunoblot analysis with anti-GFP antibody of a pre1-1
pre2-2 mutant (WCG4-11/22a) or a congenic PRE1 PRE2
wild type strain (WCG4a) transformed with GFPuv and GFPuv-mODC
expression vectors. Extracts from mutant and wild type cells were
prepared and analyzed in parallel and immunoblots were identically
exposed.
4 and
5
subunits of the 20 S core particle and shows marked defects in the
degradation of proteasome substrates (24). The GFP-mODC fusion protein
accumulated to a significantly higher steady-state level in the
pre1-1 pre2-2 mutant when compared with the congenic wild
type strain, whereas the abundance of the GFP control was unchanged
(Fig. 2D). In summary, these results demonstrate that, as in
mammalian cells, the COOH terminus of mODC is sufficient to confer
proteasome-mediated proteolysis on an otherwise stable protein in
S. cerevisiae, and that Cys441 is critical for
this function.
spe2
mutant (NH2-terminal
epitope tags interfere with expression and regulation of yODC
42).4 The
spe1
spe2
mutant, by precluding polyamine
biosynthesis, allows manipulation of cellular polyamine pools by
addition of exogenous spermidine. The addition of excess exogenous
spermidine had no effect on the steady-state level of
FLAGmODC protein (Fig.
3A) or its activity (data not
shown). Measurements of FLAGmODC turnover by
pulse-chase analysis in the presence or absence of exogenous spermidine
confirmed that polyamines did not accelerate the degradation of
FLAGmODC in yeast cells (Fig. 3B). In contrast,
excess polyamine administration greatly reduced the abundance of
yODCFLAG (Fig. 3A), indicating that the yODC
regulatory factor was indeed induced, and that the epitope tag had no
effect on polyamine regulation of yODC. We conclude that the endogenous
yODC regulatory system does not act on mODC.
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Fig. 3.
Polyamines do not regulate mODC stability in
yeast cells. A, immunoblot analysis with anti-FLAG
antibody of spe1 spe2
cells transformed with
p414ADH vectors expressing wild type FLAGmODC or
yODCFLAG. Transformants were maintained in medium with 0.1 µM spermidine (
SPD) or treated with 1.0 mM spermidine (+SPD). B, pulse-chase
analysis of FLAGmODC turnover in
spe1
spe2
transformants. Cultures were
supplemented with spermidine as described in A, or left
untreated, and FLAGmODC was immunoprecipitated with
anti-FLAG affinity gel.
T) obviated the polyamine-induced frameshifting otherwise
required for the translation of AZ1. In initial experiments we
expressed the AZ1
T ORF from multicopy vectors with promoters of
various strengths (33) in yeast transformants also expressing
FLAGmODC from a multicopy vector (Fig.
4A). The steady-state levels of FLAGmODC in these transformants exhibited a modest
decrease in response to expression of AZ1 from vectors with increasing
promoter strength. This decrease was presumably because of increased
levels of AZ1 expression, an assumption corroborated by the almost
complete inhibition of ODC enzymatic activity in the transformants also expressing AZ1 from stronger promoters (data not shown). To confirm AZ1
expression directly, we appended a single copy of the FLAG epitope to
the NH2 terminus of AZ1
T, and expressed this ORF
(FLAGAZ1
T) and FLAGmODC from centromeric
yeast expression vectors. We compared the steady-state level expression
of FLAGmODC from the ADH promoter in yeast
transformants also carrying FLAGAZ1
T expressed from the
stronger GPD promoter, or an empty vector. In agreement with
the previous result, expression of FLAGAZ1 reduced the
steady-state level of FLAGmODC compared with the empty
vector control (Fig. 4B).
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Fig. 4.
AZ1 accelerates mODC degradation in yeast
cells. A, immunoblot analysis with anti-FLAG antibody
of spe1 spe2
cells transformed with a
p424ADH vector expressing wild type FLAGmODC, and an empty
vector (p426GPD, lane 1) or untagged AZ1 expressed from
p426ADH (lane 2), p426TEF (lane 3), or p426GPD
(lane 4) expression vectors. A nonspecific, cross-reacting
protein (*) is shown as a loading control. B, immunoblot
analysis with anti-FLAG antibody of
spe1
spe2
cells transformed with a p414ADH
vector expressing wild type FLAGmODC and an empty vector
(p416GPD) or FLAGAZ1 expressed from p416GPD. C,
pulse-chase analysis using anti-FLAG affinity gel for
immunopurification of labeled FLAGmODC from the
transformants described in B. D, quantitation of
FLAGmODC turnover data shown in C. Transformants
co-expressed with FLAGmODC and FLAGAZ1
(open squares) or an empty vector control (filled
squares).
-galactosidase and Ub or
HAUb were similarly analyzed in parallel.
Leu-
-galactosidase is a substrate of the N-end rule-mediated
ubiquitinylation pathway, and is rapidly degraded in yeast cells
because of the presence of a destabilizing Leu residue at its
NH2 terminus (43). We expressed both FLAGmODC
and Leu-
-galactosidase from vectors bearing the ADH
promoter. To further enhance the detection of transient
polyubiquitinylated forms, we utilized a proteasome inhibitor and
carried out expression in a strain deleted for RPN4, a
transcriptional activator of genes encoding proteasomal subunits. We
confirmed a previous report (44) that cells with an rpn4
allele, unlike cells with wild type RPN4, are sensitive to
the effects of proteasome inhibitors (data not shown). Proteasome
function in these transformants was inhibited by treatment with 100 µM MG132, a peptide aldehyde inhibitor of the proteasome
(45). In cells expressing both Leu-
-galactosidase and
HAUb, HAUb-Leu-
-galactosidase conjugates
were detectable with anti-HA antibodies following immunoprecipitation
with anti-
-galactosidase antibody (Fig.
5, lane 6). Ub conjugates were
not detectable with anti-HA antibody following immunoprecipitation if
an untagged form of Ub was co-expressed with Leu-
-galactosidase
(lane 5). In contrast, following co-expression of
FLAGmODC with HAUb, no HAUb
conjugates were detectable in association with mODC (lane
4). Some very high molecular weight material stained faintly with the anti-HA antibody in the transformant expressing
FLAGmODC and HAUb following immunoprecipitation
with anti-FLAG antibody (lane 4), but similar staining was
seen when the anti-
-galactosidase antibody was used (as a control
for nonspecific immunoprecipitation) in a transformant expressing
FLAGmODC and HAUb (lane 2),
providing evidence that this material was not specifically associated
with FLAGmODC. A similar experiment was performed in an
RPN4 wild type strain without use of a proteasome inhibitor
and yielded similar results (data not shown). In summary,
ubiquitinylated forms of FLAGmODC were not detectable in
yeast transformants under conditions that readily supported detection
of conjugated forms of a control protein.
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Fig. 5.
Ub-mODC conjugates are not detectable in
yeast cells. Vectors (Yep96 or Yep112 (59)) bearing Ub or
HAUb expressed from the Cu2+-inducible
CUP1 promoter were co-transformed into MHY74
rpn4 mutant cells with either p415ADH vectors expressing
FLAGmODC (lanes 1-4) or Leu-
-galactosidase
(lanes 5 and 6). Following induction with 100 µM CuSO4 and inhibition of
proteasome-specific proteolysis with 100 µM MG132, cell
extracts were immunoprecipitated (IP) with the indicated
antibody, fractionated by SDS-7.5% PAGE, transferred to
nitrocellulose, and probed with anti-HA-horseradish peroxidase
conjugates. The positions of FLAGmODC and
Leu-
-galactosidase following fractionation by SDS-7.5% PAGE are
indicated.
mutants cellular
ubiquitin levels are severely depleted (48). We compared the
degradation of FLAGmODC and Leu-
-galactosidase in
uba1-2 and doa4
mutants, and a congenic wild
type strain. In both mutants, pulse-chase analysis demonstrated that
the ubiquitin-dependent substrate, Leu-
-galactosidase, was stabilized (Fig. 6, middle
panels). However, there was no apparent effect on the degradation
of FLAGmODC in these mutants compared with wild type (Fig.
6, left panels).
View larger version (38K):
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Fig. 6.
Mouse ODC degradation is not impaired in
yeast mutants defective for ubiquitin metabolism. Pulse-chase
analysis of FLAGmODC, yODCFLAG, and
Leu- -galactosidase expressed in MHY501 (wild type), MHY1409
(uba1-2), or MHY623 (doa4
) transformants.
FLAG-tagged mODC and yODC were expressed from p414ADH and p414SPE1
vectors, respectively, and immunoprecipitated with anti-FLAG affinity
gel. Leu-
-galactosidase was expressed from p415ADH, and
immunoprecipitated with anti-
-galactosidase antibody. Transformants
expressing yODCFLAG were treated with 1 mM
spermidine. The positions of the relevant band in each panel are marked
(*).
mutants and the congenic wild type strain by
pulse-chase analysis following treatment with excess spermidine (Fig.
6, right panels). This analysis indicated that
yODCFLAG was degraded with similar kinetics in wild type
and mutant strains. We also found that polyamine regulation of
endogenous yODC activity in these mutants was indistinguishable from
that of the congenic wild type strain (data not shown). These results
suggest that both the constitutive rapid degradation of mODC and the
polyamine-induced degradation of endogenous yODC occur independently of
ubiquitinylation in S. cerevisiae.
subunit of the 20 S
core particle. We prepared agarose beads loaded with either the 26 S
holoenzyme or 20 S core particle of the proteasome. We verified the
composition of the immunopurified complexes by SDS-PAGE of a portion of
the preparations eluted under denaturing conditions or following
elution with FLAG peptide (Fig.
7A). The proteins visualized
following Coomassie staining of these gels showed the expected patterns
for 20 S and 26 S proteasomes (28, 49).
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Fig. 7.
Mouse ODC is degraded by purified yeast
proteasomes. A, SDS-PAGE of proteasome preparations
eluted from anti-FLAG affinity gel by boiling in SDS loading buffer
(denaturing elution) or by incubation with 100 µg/ml FLAG peptide
(peptide elution). MW, molecular weight markers (kDa); Ig H
and L chain eluted by buffer. B, effects of ATP and
proteasome composition on mODC degradation in a purified degradation
system. Degradation assays were carried out using equivalent amounts
(100 nM) of the proteasome holoenzyme (26 S), containing
both the 20 S core particle and 19 S regulator, or the 20 S core
particle alone (20S). Reactions were carried out at 37 °C
for 30 min in the presence of 1 mM ATP (filled
bars), or following ATP depletion by washing proteasomes affixed
to the affinity matrix in reaction buffer lacking ATP (open
bars). C, effects of ODC COOH-terminal mutants on the
degradation of mODC by yeast 26 S (open bars) or rat liver
26 S (filled bars) proteasomes in an in vitro
degradation system. Degradation assays were carried out as in
B using radiolabeled wild type mODC (wild type),
or mODC lacking its 5 COOH-terminal amino acids (ODC1-456),
or containing the C441S mutation (ODC C441S). D,
effects of AZ1 on the in vitro degradation of 50 nM mODC by yeast 26 S or rat liver 26 S proteasomes in the
absence (open bars) or presence (filled bars) of
400 nM AZ1.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Hui Chen and Frieder Merz for constructing plasmids used in this study, and Robert Swanson (University of Chicago, Chicago, IL), Mark Hochstrasser (Yale University, New Haven, CT), and Rati Verma and Raymond Deshaies (California Institute of Technology, Pasadena, CA) for strains, plasmids, and helpful advice.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Grant GM45335 from the National Institutes of Health (to P. C.).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.
The on-line version of this article (available at
http://www.jbc.org) contains Fig. 1.
Supported by National Research Service Award Postdoctoral
Fellowship GM20527 from the National Institutes of Health.
§ To whom correspondence should be addressed: Dept. of Microbiology and Immunology, University of California, San Francisco, CA 94143-0414. Tel.: 415-476-1783; Fax: 415-476-8201; E-mail: pcoffin@itsa.ucsf.edu.
Published, JBC Papers in Press, January 31, 2003, DOI 10.1074/jbc.M211802200
2 M. A. Hoyt and P. Coffino, unpublished results.
3 M. Zhang, C. M. Pickart, and P. Coffino, EMBO J., in press.
4 F. Merz and P. Coffino, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: ODC, ornithine decarboxylase; AZ1, antizyme 1; GFP, Aequorea victoria green fluorescent protein; HA, hemagglutinin; mODC, Mus musculus ornithine decarboxylase; ORF, open reading frame; yODC, Saccharomyces cerevisiae ornithine decarboxylase; E1, ubiquitin-activating enzyme; Ub, ubiquitin.
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