Analysis of Polyubiquitin Conjugates Reveals That the Rpn10 Substrate Receptor Contributes to the Turnover of Multiple Proteasome Targets

EXPERIMENTAL PROCEDURES

Yeast Strains and Plasmids—

All S. cerevisiae strains used in this study are listed in supplemental Table 1. RJD2997 was generated by integrating the plasmid RDB1848, which contains the coding sequences for H₆-ubiquitin flanked by the GPD constitutive promoter and PGK terminator sequences (18), into the TRP1 locus. Control strain RJD2998 was obtained by integrating the empty vector into the TRP1 locus. Mutant rpn10Δ was retrieved from the Yeast Deletion Library (Open Biosystem) and back-crossed into the W303 background. Gcn4-Myc9 was previously described (19). S288C strains with TAP-tagged genes were retrieved from the Yeast TAP-Fusion Library (Open Biosystem).

The H₈-ubiquitin coding sequence was placed between the GPD constitutive promoter and PGK terminator sequences in pRS316 (RDB1851). A pair of primers (5′-GCGGATCCATGAGAGGTAGTCACCACCATCATCACCATCATCACGGTGGTATGCAGATTTTCG-3′ and 5′-GAGCTCGAGACCACCTCTTAGCCTTAGCAC-3′) was used to amplify by PCR yeast ubiquitin (the first repeat of the UBI4 locus). The PCR fragment was digested with BamHI and XhoI and ligated into the yeast expression vector pG-1 (20) (digested with BamHI and SalI). An EcoRI-NaeI fragment containing H₈-ubiquitin was then ligated into pRS316 (digested with EcoRI and SmaI).

Immobilization of Polyubiquitin Binding Proteins—

GST-Rad23 and GST-Dsk2 were generous gifts from H. Kobayashi and H. Yokosawa, respectively. Fusion proteins were expressed in BL21(DE3)/pLysS and purified using glutathione-Sepharose resin. Ten mg of GST-Dsk2p and 20 mg of GST-Rad23 were separately coupled to 1.5 ml of resin volume of CNBr-activated Sepharose 4B (Amersham Biosciences) in 100 mm NaHCO₃, pH 8.3, 0.5 m NaCl. Coupled resin was stored at 4 °C in a 50% slurry with 100 mm Tris-HCl, pH 8.0, 0.5 m NaCl, 0.02% NaN₃.

Two-step Purification—

Cells were grown in 6 liters of YPD medium (2% peptone, 1% yeast extract, 2% dextrose) at 25 °C to an A₆₀₀ of 1.5. Cells were washed with 1/6 volume of ice-cold TBS followed by 1/30 volume of ice-cold TBS with 1 mm 1,10-phenanthroline, 10 mm iodoacetamide. Cells were lysed using a One Shot Cell Disrupter (Constant Systems) at 30,000 psi in 40 ml of lysis buffer (300 mm NaCl, 0.5% Triton X-100, 50 mm sodium phosphate, pH 8.0, 0.5 mm AEBSF, 5 μg/ml aprotinin, 5 μg/ml chymostatin, 5 μg/ml leupeptin, 1 μg/ml pepstatin A, 1 mm 1,10-phenanthroline, 10 mm iodoacetamide). Lysate (typically 1.5 g of protein) was cleared by centrifugation at 4 °C in a Sorvall SS34 for 20 min at 14,000 rpm. Two milligrams each of GST-Rad23 and GST-Dsk2 coupled to Sepharose (pre-equilibrated with lysis buffer) were added to the clarified lysate and mixed for 90 min at 4 °C. The resin was then washed with 40 ml of lysis buffer, further mixed for 15 min with 20 ml of 50 mm sodium phosphate, pH 8.0, 2 m NaCl, and washed once with 20 ml of 50 mm sodium phosphate, pH 8.0, 2 m NaCl and twice with 20 ml of 50 mm sodium phosphate, pH 8.0, 300 mm NaCl, 0.1% Triton X-100. Elution was performed at room temperature with two successive incubations with 1 ml of urea buffer (UB: 8 m urea, 100 mm NaH₂PO₄, 10 mm Tris-HCl, pH 8.0), and imidazole was added to a final concentration of 20 mm. Eluate was then mixed with 125 μl of nickel magnetic bead slurry (Promega V8565, prewashed in the UB) for 60 min on a rotating wheel. The beads were washed with 1 ml of UB and mixed for 15 min with UB supplemented with 0.5% SDS. The beads were then washed with 1 ml of UB with 0.5% Triton X-100 and mixed for 15 min with another 1 ml of UB with 0.5% Triton X-100. The last procedure was repeated using UB only.

To generate peptides for MS-based sequencing, we performed the tryptic digest directly on the beads. The beads were incubated with 500 μl of UB with 3 mm Tris-(2-carboxyethyl)phosphine (T-CEP) for 20 min and then for another 15 min following addition of iodoacetimide to 11 mm. The buffer volume was reduced to 75 μl by removing excess liquid, and 0.2 μg of endoproteinase Lys-C (Roche) was added. Beads were incubated at 37 °C with intermittent shaking for 5 h. Dilution buffer (225 μl of 100 mm Tris-HCl, pH 8.0, 1.33 mm CaCl₂) was then added followed by 1 μg of trypsin (Roche Applied Science), and the beads were further incubated with intermittent shaking for 16 h at 37 °C. The supernatant was carefully collected, and formic acid was added to a final concentration of 5%.

MS and Data Analysis—

The proteolytically digested sample was further processed for multidimensional chromatography coupled in-line to ESI-MS as described previously (21). As a variation to the chromatography program, samples were stepped off the strong cation exchanger phase of the triphasic column using 12.5, 20, 30, 40, and 100% buffer C (500 mm ammonium acetate, 5% ACN, 0.1% formic acid). Centroided fragmentation spectra acquired by Xcalibur 1.3 (ThermoElectron) were evaluated for spectrum quality and charge state using 2to3 (22) and searched against the translated open reading frames of the Saccharomyces Genome Database (SGD (23); release time stamp: 07/26/2004; 6,860 entries) with Sequest (version 27, revision 9; Ref. 24) utilizing unified input and output files (25). Relevant Sequest parameters used were: (i) peptide mass tolerance of 3.0 amu, (ii) parent ion masses were treated as monoisotopic, (iii) fragmentation ion masses were treated as averaged, and (iv) a 57.0-amu static modification on cysteines accounted for alkylation. Sequest results were filtered using DTASelect 1.9 and Contrast (26) with the following requirements for peptide and locus identifications considered valid: minimum Xcorrs of 1.8, 2.5, and 3.5 for singly, doubly, and triply charged ions, respectively; a minimum ΔCn of 0.08; and a minimum of two valid peptides per locus.

Small Scale Cell Extraction, IMAC, and Western Blotting—

For direct comparison of protein level in wild-type and rpn10Δ strains, S288C cells were grown in YPD at 25 °C until an A₆₀₀ of 0.5–1 was reached. An amount of yeast cells corresponding to 4–5 A₆₀₀ was collected, briefly washed with 1 ml of 1× TBS, and frozen in liquid nitrogen. Cells were directly resuspended in prewarmed sample buffer, incubated for 2 min at 96 °C, lysed with glass beads in a FastPrep 120 (Thermo Savant) for 45 s with a speed setting of 5.5, and incubated for another 4 min at 96 °C. For IMAC purification of H₈-ubiquitin, cells transformed with a URA3-based plasmid coding for H₈-ubiquitin were grown in 100 ml of SD-URA medium (0.67% yeast nitrogen base, 5% dextrose) at 30 °C to an A₆₀₀ of 1. TCA (20% final) was added directly to the cell culture, and cells were incubated for 10 min on ice and washed with ice-cold 100 mm Tris-HCl (once with pH 8.5, twice with pH 8.0). Cells were resuspended in 0.6 ml of 0.2% SDS, 8 m urea, 100 mm Hepes, pH 8.0, 1 mm 1,10-phenanthroline, 5 mm N-ethylmaleimide (NEM), 0.5 mm AEBSF, 5 μg/ml aprotinin, 5 μg/ml chymostatin, 5 μg/ml leupeptin, 1 μg/ml pepstatin A, and lysed by agitation with glass beads in a FastPrep 120. Glass beads were further washed with 0.6 ml of lysis buffer without SDS, and lysate (containing 0.1% SDS) was cleared 10 min at 14,000 rpm in a microcentrifuge. Imidazole (20 mm final) and nickel magnetic beads (70 μl) were added to 8.5 mg of lysate protein and mixed for 1 h at room temperature. Beads were then washed three times in 0.1% SDS, 8 m urea, 100 mm Hepes, pH 8.0, and proteins were eluted in SDS-PAGE sample buffer supplemented with 1 m imidazole, 4 m urea, 50 mm Hepes, pH 8.0. TAP-tagged proteins were detected using the anti-calmodulin binding peptide antibody (Upstate Biotechnology), ubiquitin with MAB1510 (Chemicon International), Cdc28 with PSTAIR antibody (Santa Cruz Biotechnology), and Gcn4-Myc9 with 9E10 monoclonal antibody.

RESULTS

Two-step Purification of Ubiquitin Conjugates—

We performed two-step purification of ubiquitin conjugates (Fig. 1) from cells that express ubiquitin fused to an amino-terminal hexahistidine tag (H₆-ubiquitin), and as control we repeated the procedure with cells that do not express H₆-ubiquitin. In both experiments, the first purification step yielded a similar amount of proteins, whereas the IMAC only recovered appreciable material from the H₆-ubiquitin strain (Fig. 2A). The signal revealed by silver staining of material fractionated by SDS-PAGE ranged from 50 to 250 kDa and produced a spread rather than discrete bands, as expected for a large collection of different proteins conjugated to ubiquitin chains of various lengths. We calculated that the first step in purification recovered about 15% of the polyubiquitin conjugates in the cell (Fig. 2B). Notably, mono-, di-, and triubiquitin species were not recovered. This implies that the UBA domains of Rad23 and Dsk2 were only enriching for proteins conjugated to ubiquitin chains that contained more than three ubiquitins. Because a tetraubiquitin chain is thought to comprise the minimal signal for targeting substrates to the proteasome for degradation (27, 28), the UBA affinity step appears to enrich specifically for those ubiquitin conjugates that are proteasome substrates. In the second step, the majority of the ubiquitin conjugates (>80%) eluted from the first resin were recovered (Fig. 2C). In this experiment, only 25–30% of the bound material was eluted with sample buffer from the nickel beads (data not shown). Overall, our procedure resulted in a 3,000- to 5,000-fold enrichment of polyubiquitin conjugates (1,500 mg of protein extract resulted in 30–50 μg of protein, representing 10% of the polyubiquitin in the cell).

MS Analysis—

Purified proteins were directly digested on the nickel beads, and the peptide mixture was analyzed by multidimensional LC-MS/MS or MudPIT. Sequest and DTASelect algorithms were used to analyze the spectra generated by the complex mixture of affinity-purified proteins, and 180 nonredundant proteins were identified (supplemental Table 2). The most abundant protein in our analysis was ubiquitin. Of a total of 5,347 sequencing events, 457 peptides derived from ubiquitin. This was expected because ubiquitin should be the most prominent protein after the purification. For clarity, we further filtered our data by removing transposon-related genes, duplicated genes, ubiquitin fusion genes, and Rad23 and Dsk2 that leached from the resin used in the first purification step (data not shown). The 127 remaining proteins are listed in Table I. We classified these proteins in different categories according to their function (Fig. 3A). The majority of identified proteins is involved in metabolism and translation. Several proteins are components of regulated pathways, and several were previously shown to be targets for degradation. These include Ole1, a short lived protein (29), Rpo21, which is ubiquitylated by Rsp5 (30), and Gdh1 and Mdh2, which were shown previously to be targeted for proteolysis (31, 32). Moreover, the list includes proteins for which ubiquitylation sites were previously identified; 14 of our 127 proteins were among the 71 identified in the initial global study of ubiquitylated proteins (33), and 8 of our 127 proteins were among the 33 found in a screen for membrane-associated ubiquitylated proteins (34). Thus, although we identified only ∼2% of the yeast proteome (127/∼6,000), these proteins accounted for 21% of the ubiquitylated proteins identified by Gygi and co-workers (33, 34), a 10-fold enrichment.

Because our ultimate goal was to compare the pool of ubiquitylated proteins in wild-type and rpn10Δ cells, it was important to assess the variability of the MS analysis. The sample from the two-step purification described above had been split in half after the trypsin digest but prior to the MS analysis. When the second half of the sample was analyzed, we identified 176 proteins (supplemental Table 3). The two LC/LC-MS/MS analyses of the same sample were then compared using the Contrast algorithm (Fig. 3B). More than 80% of the proteins identified in one analysis were found in the other analysis. We noticed that the variability was accounted for mainly by proteins identified by two peptides (as the loss of one peptide identification for a particular protein led to its exclusion from the analysis). When we also took into account proteins identified by only one peptide, ∼95% of proteins identified by two peptides in either dataset were also identified by at least one peptide in the duplicate analysis (Fig. 3B). This indicated that there was some variation in the data analysis, albeit tolerable, arising from either the HPLC or mass spectrometer. Moreover, proteins defined by our minimum cutoff of two peptides (and thus possibly of low abundance in the purified sample) were disproportionately susceptible to being overlooked. Because many potential targets of interest might be in the inabundant category, we decided to perform our subsequent analyses in triplicate to ensure the identification of a maximum number of ubiquitin conjugates.

Impact of the Proteasome Substrate Receptor Rpn10 on the Pool of Ubiquitin Conjugates—

Our key motivation for developing proteomic methods to identify ubiquitin conjugates on a global scale was to use the method to identify substrates/targets for ubiquitin ligase and isopeptidase enzymes and other specificity determining factors in the UPS. In particular, we sought to determine the breadth of the impact of Rpn10 on ubiquitin-dependent proteolysis. We reasoned that deletion of RPN10 would prevent the degradation of substrates dependent on Rpn10 for turnover. These substrates would then accumulate as polyubiquitylated conjugates. To proceed, we collected and analyzed six independent samples; three were obtained from wild-type cells (supplemental Tables 2 and 4) and three others from rpn10Δ cells (supplemental Table 5). We compared the six datasets using the Contrast algorithm (Fig. 3C). The variability between the different datasets (∼30%) was in general higher than previously observed between two identical samples (Fig. 3B). This was expected because it is essentially impossible to grow cells, lyse cells, and carry out consecutive affinity purification steps in a manner that is perfectly precise. Nevertheless, to identify the candidate targets of Rpn10, proteins represented in any of three rpn10Δ samples but not in any RPN10 sample were extracted and rank-ordered according to sequence coverage of the identified protein (Table II). What is particularly noteworthy is that the second highest ranked candidate in this subtractive screen of the entire S. cerevisiae proteome was the cell cycle regulator Sic1, which is ubiquitylated by the SCF^Cdc4 complex at the G1/S transition (35). We had previously shown that Sic1 degradation is substantially dependent upon Rpn10 (17), suggesting that it is likely to accumulate as a ubiquitylated species in rpn10Δ cells (an assumption that was not addressed previously but has been validated as described below). Other candidates revealed by this substractive approach are also known to be targets for ubiquitylation. The transcription factor Gcn4 is targeted for proteolysis after ubiquitylation by SCF^Cdc4 complex (19, 36, 37), and Aro10, Ald6, Erg3, and Ecm21 were identified as ubiquitylated proteins in a global analysis (33). Taken together, these findings suggest that our subtractive approach was sufficiently sensitive to identify critical regulatory targets of the UPS, even those of exceptionally low abundance such as Gcn4, which is estimated to be present at less than 50 molecules per cell (38).

Validation of Rpn10 Targets—

To evaluate the role of Rpn10 in turnover of candidate substrates identified by our MudPIT approach, we assayed several of the proteins from Table II for abundance and ubiquitylation. First, we compared protein levels in RPN10 and rpn10Δ strains in which the endogenous loci were modified to encode the candidate proteins with TAP tags fused to their C termini (Fig. 4A and Table II). For several candidates, protein levels were elevated in the rpn10Δ strain, suggesting that normal turnover of these proteins was Rpn10 dependent. For Gcn4, we employed a well characterized allele that encodes a carboxyl-terminal Myc9 tag integrated into the GCN4 locus (19). We found that Gcn4 accumulated in rpn10Δ extracts, and we could also detect species migrating with a lower mobility that correspond to polyubiquitylated Gcn4 (Fig. 4B).

In addition to inabundant proteins like Gcn4, our analysis also identified highly abundant proteins as such the ribosomal subunit Rpl13B. However, by Western blotting we could not see any increase in the level of Rpl13B in rpn10Δ (data not shown). We reasoned that in this case perhaps only a small fraction of the protein pool was targeted for degradation, and thus the overall protein abundance was not altered in rpn10Δ. To test this, we devised a single-step purification with nickel beads using cells transformed with a plasmid that expressed ubiquitin with an octahistidine tag fused to the amino terminus (H₈-ubiquitin). Purified proteins were detected with the TAP tag antibody. An untagged rpn10Δ strain that expressed H₈-ubiquitin was used as a negative control and gave no signal in the Western blot (Fig. 4C). After performing the same procedure with a Sic1-TAP strain, we noticed the distinctive accumulation of high molecular mass Sic1 conjugates in rpn10Δ but not in RPN10 cells that expressed H₈-ubiquitin (Fig. 4C). No signal was readily detected rpn10Δ cells not expressing the H₈-ubiquitin. Therefore, ubiquitylated Sic1 specifically accumulated in cells lacking Rpn10. Rpl13B showed similar behavior. Although there was some nonspecific binding of unmodified Rpl13B to the nickel beads (lower band present in all three lanes), Rpl13B species that migrated at high molecular masses (>250 kDa) were exclusively detected in rpn10Δ cells that expressed H₈-ubiquitin. Notably, species modified with one, two, and three ubiquitins were also detected in wild-type cells whenever H₈-ubiquitin was expressed. However Rpl13B was only detected by MS in samples from rpn10Δ cells. Therefore the species modified with one, two, and three ubiquitins that were also present in RPN10 cells most likely were not enriched in the two-step purification, as is the case for free (i.e. not substrate-linked) mono-, di-, and triubiquitin (Fig. 2B). Vhs2 protein level was also found unaltered in rpn10Δ cells (despite its relative low abundance), but ubiquitylated Vhs2 was detected after IMAC of extracts from rpn10Δ cells that expressed H₈-ubiquitin (Fig. 4C).

DISCUSSION

In this article, we describe a new approach to the purification and analysis of ubiquitin conjugates in the budding yeast S. cerevisiae. Our approach involves two affinity purification steps. The first step selects for ubiquitin chains that were able to bind recombinant UBA domain-containing proteins and thus were most likely competent to support degradation of attached proteins. In the second step, ubiquitin conjugates that contain H₆-ubiquitin were enriched by IMAC. Conjugates that survived the two enrichment steps were digested to yield peptides, which were separated by multidimensional chromatography and sequenced by MS/MS. This protocol enabled us to identify a collection of candidate ubiquitin-conjugated proteasome substrates. By performing a “subtractive” comparison of conjugates recovered from wild-type cells versus rpn10Δ cells that lack the proteasome substrate receptor Rpn10, we were able to identify a collection of proteins that accumulate selectively in rpn10Δ and thus are candidate ligands for Rpn10. This effort revealed that the pool of candidate Rpn10 ligands is much larger than appreciated previously from one-off analyses.

The approach described here differs from prior “proteome-wide” analyses of ubiquitin- (33, 34) and SUMO-conjugated proteins (39–44) in several important respects. First, we present data on replicate analyses. We found modest variation (∼17%) in duplicate MS analyses of a single sample, but significant variations (∼30%) when the entire affinity purification and LC/LC-MS/MS analysis were repeated. Performing replicate analyses is thus of considerable importance when comparing the ubiquitin conjugate proteome in different strains (e.g. wild type and rpn10Δ) to ensure that any differences seen are due to the mutation under study and are not simply a product of experimental variability. Performing replicate experiments also helps to ensure that an analysis is as thorough as possible. For example, some candidates that were validated (e.g. Sic1) were only identified in one of three analyses. Indeed, of the candidates for which identification was least robust (Mlp1, Psk2, and Mdn1, each of which was found in only one analysis at <2% sequence coverage), all three were validated as being responsive to Rpn10 function. Thus, we strongly recommend that multidimensional analyses be performed with replicate samples both to minimize false positives and to enhance identification of target proteins.

A second key difference is that we employed a “functional” affinity purification step in tandem with a tag-dependent affinity purification step. By comparison, Gygi and co-workers (33, 34) employed a single nickel-nitrilotriacetic acid affinity purification step in their analyses of the ubiquitin proteome. The inclusion of a second, function-based affinity step had two important consequences; first, it enabled superior enrichment for ubiquitin-conjugated proteins, and second, it focused our analysis on a particular subset of ubiquitin-conjugated proteins (i.e. those that are candidate substrates for the proteasome). In our hands, single-step purification with H₆-ubiquitin led to a relatively modest enrichment of ubiquitin conjugates (100- to 200-fold)² as compared with the two-step purification (3,000- to 5,000-fold). This is in keeping with our experience that ∼0.5% of total yeast extract proteins bind specifically to IMAC resins. Thus, it is possible that a fair fraction of the proteins identified previously are not bona fide UPS substrates. Importantly, our approach has permitted the identification of even the extremely inabundant UPS substrate Gcn4, which is present at less than 50 molecules per cell (38). Consistent with the greater degree of target focus intrinsic to our analysis, we did not identify proteins that are known to be conjugated with a single ubiquitin (e.g. histone H2A, B), nor did we enrich for mono-, di-, or triubiquitin chains (Fig. 2B). Finally, when we searched for peptides of ubiquitin itself that carried the Gly-Gly signature, Lys⁴⁸ was found to be the most prominent conjugation site that was recovered (data not shown). Lys²⁹, Lys³³, and Lys⁶ were more rarely identified, and modified Lys⁶³ was not found. These findings suggest that we have established a new approach to identify specifically those proteins that are polyubiquitylated substrates of the proteasome. In the future, other ubiquitin receptors, like proteins containing UIM domains that bind mono-ubiquitylated targets in the endocytic pathway (e.g. Vps27 and Ent1) or ZnF domains that bind selectively to Lys⁶³-linked ubiquitin chains (45), may be used to identify factors in nonproteasomal pathways that are regulated allosterically by ubiquitylation.

Of the more than 120 proteins that we implicated as substrates of the UPS, most function in translational and metabolic pathways, and half of the candidates have high codon adaptation index values (>0.4).³ Many ribosomal proteins were identified including some that were shown previously to be ubiquitylated, like Rpl28, Rps3, and Rps20 (33, 46). Because ribosomes are highly abundant and formed by tight macromolecular interactions, we cannot exclude that some of the identified proteins were contaminants. However, it is also possible that some of these candidate substrates might represent biosynthetic intermediates that fail to fold or assemble properly, resulting in their rapid degradation either during or shortly following the completion of translation (47, 48). In the latter case, one would predict that the UPS might have little impact on the total level of the candidate protein and that only a very small fraction of the total protein pool in the cell is ubiquitylated (depending on the fraction of the protein that misfolds or misassembles). This is exactly what we observed for Rpl13B. If a small fraction of Rpl13B fails to assemble properly and is degraded rapidly by the UPS, it could help to explain the presence of many proteins with high codon adaptation index values in our analysis. Thus, the bulk of proteins degraded by the proteasome in yeast cells might correspond to misfolded, damaged, or improperly translated proteins rather than proteins such as cyclins, CDK inhibitors, and transcription factors whose functions are regulated by proteolysis. Further studies will be required to address the important issue of substrate flux through the UPS in yeast. Notably, our method provides a means to identify substrates of the chaperone pathways that enable efficient protein folding and assembly as well as the ubiquitin ligases that target misfolded proteins for degradation by the UPS.

To gain a sense of the quality of our subtractive dataset of conjugates uniquely found in rpn10Δ samples, we employed two different assays to evaluate 17 of the 54 candidate Rpn10 substrates. The first and simplest assay was to compare by immunoblotting the level of the candidate protein in wild-type and rpn10Δ cells on the assumption that Rpn10 substrates might accumulate to a higher level in rpn10Δ. However, we recognized that there may be substrates for which only a small fraction of the total pool is degraded by an Rpn10-dependent pathway, and these substrates might fail this test. Thus, we devised a second assay that measured the level of ubiquitylated candidate protein that was present in wild-type and rpn10Δ cells. This second assay allowed us to confirm some candidate proteins (e.g. Rpl13) that were not validated by the first assay. Ultimately, we were able to confirm that nearly 60% (10 of 17) of the candidates analyzed are responsive to Rpn10 function. It is important to note that the validation experiments were done with TAP-tagged chromosomal loci (which are in the S288C genetic background), and that the cells were grown in synthetic medium to select for a H₈-ubiquitin expression plasmid. By contrast, the affinity purification-mass spec analyses were performed with cells of the W303 strain background grown in rich (YPD) medium. Thus, a failure to confirm a candidate should not be construed as definitive evidence that the candidate is not an Rpn10 ligand. Nevertheless, the apparent high rate of false positives underscores that it is critical to carry out secondary analyses to confirm data acquired in multidimensional MS analyses. Future developments, including the implementation of quantification methods and higher stringency biochemical separations, may reduce the experimental variations and false positive rate.

A previous study (17) from this laboratory revealed that the proteasome substrate receptors Rpn10 and Rad23 can promote degradation of specific subsets of UPS targets and suggested that Rpn10 targets might be restricted to a small class of UPS substrates. However, that study was based on the piecemeal examination of a handful of UPS targets, and it was not designed to reveal the full spectrum of substrates targeted to the proteasome by a given ubiquitin chain receptor. By using the two-step purification multidimensional MS method described here, we have identified several dozen candidate ligands for an Rpn10-dependent targeting pathway that function in a broad range of processes including metabolism, transcription, translation, nuclear transport, and cell cycle. By applying this approach to mutants lacking other receptors (e.g. rad23Δ, dsk2Δ), it should be feasible to begin the task of constructing a “linkage map” that reveals the spectrum of substrates that are targeted to the proteasome by a specific receptor, which may in turn provide insight into the mechanisms that underlie the allocation of ubiquitylated substrates to different receptor pathways.