Ubiquitin-independent Mechanisms of Mouse Ornithine Decarboxylase Degradation Are Conserved between Mammalian and Fungal Cells*,

Martin A. HoytDagger, Mingsheng Zhang, and Philip Coffino§

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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (MATalpha his3 leu2 trp1 ura3 spe1Delta ::HisG spe2Delta ::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 (MATalpha his3-Delta 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 rpn4Delta mutant, MHY74 (MATalpha his3-Delta 200 leu2-3,112 lys2-801 trp1-1 ura3-52 rpn4Delta ::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-beta -galactosidase vectors were transformed into MHY1409 (MATalpha his3-Delta 200 leu2-3,112 lys2-801 trp1-1 ura3-52 gal2 uba1-2 (mTn URA3)) (26), MHY623 (MATalpha his3-Delta 200 leu2-3,112 lys2-801 trp1-1 ura3-52 doa4-Delta 1::LEU2) (27), or MHY501. Proteasomes were affinity-purified from strain RJD1144 (MATa his3-Delta 200 leu2-3,112 lys2-801 trp1Delta 63 ura3-52 PRE1FH::YIplac211 (URA3)) (28).

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 (mODCDelta 425-461) or Ala457 (mODCDelta 457-461). The AZ1Delta T ORF was cloned from pGEM4Z/Delta T (8) as a HindIII-EcoRI fragment into similarly digested URA3-marked Mumberg vectors.

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 -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.

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-beta -galactosidase or GFP were carried out by incubation of lysates with 1 µl of anti-beta -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% (beta -galactosidase) polyacrylamide gels. Radiolabeled proteins were visualized by autoradiography, and quantified using Scion Image software (Scion Corp.).

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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

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 spe1Delta spe2Delta 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.

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 spe1Delta spe2Delta 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 spe1Delta spe2Delta cells transformed with p414ADH vectors expressing wild type FLAGmODC or COOH-terminal mutants. B, pulse-chase analysis of FLAGmODC in spe1Delta spe2Delta 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.

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).


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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.

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 beta 4 and beta 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.

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 spe1Delta spe2Delta mutant (NH2-terminal epitope tags interfere with expression and regulation of yODC 42).4 The spe1Delta spe2Delta 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 spe1Delta spe2Delta 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 spe1Delta spe2Delta transformants. Cultures were supplemented with spermidine as described in A, or left untreated, and FLAGmODC was immunoprecipitated with anti-FLAG affinity gel.

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 (AZ1Delta T) obviated the polyamine-induced frameshifting otherwise required for the translation of AZ1. In initial experiments we expressed the AZ1Delta 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 AZ1Delta T, and expressed this ORF (FLAGAZ1Delta 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 FLAGAZ1Delta 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 spe1Delta spe2Delta 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 spe1Delta spe2Delta 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).

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-beta -galactosidase and Ub or HAUb were similarly analyzed in parallel. Leu-beta -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-beta -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 rpn4Delta 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-beta -galactosidase and HAUb, HAUb-Leu-beta -galactosidase conjugates were detectable with anti-HA antibodies following immunoprecipitation with anti-beta -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-beta -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-beta -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 rpn4Delta mutant cells with either p415ADH vectors expressing FLAGmODC (lanes 1-4) or Leu-beta -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-beta -galactosidase following fractionation by SDS-7.5% PAGE are indicated.

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 doa4Delta mutants cellular ubiquitin levels are severely depleted (48). We compared the degradation of FLAGmODC and Leu-beta -galactosidase in uba1-2 and doa4Delta mutants, and a congenic wild type strain. In both mutants, pulse-chase analysis demonstrated that the ubiquitin-dependent substrate, Leu-beta -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).


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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-beta -galactosidase expressed in MHY501 (wild type), MHY1409 (uba1-2), or MHY623 (doa4Delta ) transformants. FLAG-tagged mODC and yODC were expressed from p414ADH and p414SPE1 vectors, respectively, and immunoprecipitated with anti-FLAG affinity gel. Leu-beta -galactosidase was expressed from p415ADH, and immunoprecipitated with anti-beta -galactosidase antibody. Transformants expressing yODCFLAG were treated with 1 mM spermidine. The positions of the relevant band in each panel are marked (*).

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 doa4Delta 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.

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 beta  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.

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.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    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.

Dagger 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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ABSTRACT
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RESULTS
DISCUSSION
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