Attachment of the Ubiquitin-Related Protein Urm1p to the Antioxidant Protein Ahp1p

Strains, growth conditions, and plasmids.The yeast strains used in this study are listed in Table 1. Yeast transformations were performed as described previously (15). Haploid MATa nonessential yeast deletion strains were purchased from Research Genetics (Huntsville, Ala). Other gene deletions were constructed by PCR (4) using either the pRS (49) or pFA6a (34) plasmid series as templates. In all cases, the entire coding region was replaced with the indicated marker, and successful replacement was confirmed by PCR and phenotype when applicable.

Yeast strains were propagated using standard methods (47). Rich yeast medium containing 2% glucose (YPD) and synthetic yeast medium containing either 2% glucose (SD) or 2% galactose (SG) were prepared as described previously (47). Geneticin (Biovectra, Charlottetown, Canada) selection was performed as previously described (34). Strains were cured of URA3-containing plasmids using 5-fluoroorotic acid (Biovectra). Rapamycin (Sigma, St. Louis, Mo.) was added to the medium from a concentrated stock solution in 90% ethanol-10% Tween 20. To test for tert-butyl hydroperoxide (t-BOOH) and diamide sensitivities, cells were grown overnight in YPD medium, spotted on yeast extract-peptone-dextrose (YEPD) plates containing either 0.4 mM t-BOOH (Sigma), 1 mM t-BOOH, 1 mM diamide (Sigma), or 1.3 mM diamide. The plates were monitored after 3 days of incubation at 30°C.

The P_GAL1-driven URM1 construct that bears the N-terminal extension MTSHHHHHHMHDYKDDDDKMGS containing His₆ and FLAG tags (P_GAL1-PRS315 HFURM1) has been described (17). The other plasmids used in this study, YCpHIS3cla4-75, pML40, pML41, pRS316ADE8CLA4, pEGH P_GAL1-GST-HisX6, and pEGH P_GAL1-GST-HisX6-AHP1, were described previously (9, 35, 39, 55).

Immunoblot analysis of whole-yeast lysates and antibodies.Immunoblot analysis of yeast lysates was performed as previously described (17). When indicated, strains were treated with 1.5 mM diamide or 1.3 mM t-BOOH for 4 h prior to lysis. Urm1p was detected by using polyclonal antibody to Urm1p (17), a secondary anti-rabbit horseradish peroxidase-conjugated antibody (Bio-Rad Laboratories, Hercules, Calif.), and chemiluminescence (Pierce, Rockford, Ill.). A monoclonal antibody against Dpm1p (a kind gift from T. Stevens, University of Oregon, Eugene, Oreg.) and a secondary anti-mouse horseradish peroxidase-conjugated antibody (Bio-Rad Laboratories) were used to confirm that equal amounts of protein were loaded in each lane.

Isolation of Urm1p-conjugated proteins.SY4118 containing pRS315 P_GAL1-HFURM1 was grown at 30°C in 4 liters of YP containing 2% raffinose to an optical density at 600 nm of 0.75. Expression of P_GAL1-HFURM1 was induced by the addition of 2% galactose, and the cells were grown for an additional 4 h at 30°C. The cells were harvested by centrifugation and converted to spheroplasts by incubation with 150 μg of Zymolyase 100T (Seikagaku America, Ijamsville, Md.)/ml in 1.2 M sorbitol-50 mM potassium phosphate buffer (pH 7.5)-1 mM MgCl₂ for 45 min at 30°C. The spheroplasts were washed once with 1.2 M sorbitol and lysed in buffer A. The lysates were clarified by centrifugation at 12,000 × g, supplemented to 20 mM imidazole and 10% glycerol, and bound in batch for 1.5 h to 4 ml of Talon resin (Clontech, Palo Alto, Calif.). The Talon resin was loaded into a column, washed with 40 ml of buffer B (buffer A with 10% glycerol), and then eluted with 200 mM imidazole in buffer B. Four milliliters of the eluate was bound in batch for 1 h at 4°C to 0.4 ml of anti-FLAG agarose (Sigma). The anti-FLAG agarose was loaded into a column, washed with 50 ml of buffer B supplemented with 0.2% Trition X = 100, washed with 5 ml of buffer B, and then eluted with 1.2 ml of buffer B supplemented with 0.2 mg of FLAG peptide (Sigma). The eluate was precipitated by the addition of trichloroacetic acid to 5%, resuspended in loading buffer, and applied to a 10% acrylamide gel. Following electrophoresis, protein bands were detected by silver staining and excised. The sample was subjected to in-gel digestion (48), desalted using Millipore μC18 ZipTips, and dried. The sample was then resuspended in 5 μl of water and analyzed by liquid chromatography microspray-mass spectrometry (MS-MS) with a ThermoFinnigan LCQ DECA XP mass spectrometer (FHCRC Proteomics) using the configuration of Gatlin et al. (14). Data were collected in the data-dependent mode in which an MS scan was followed by MS-MS scans of the three most abundant ions from the preceding MS scan. The MS-MS data were searched using SEQUEST software against the translated open reading frames from the Saccharomyces Genome Database (http://genome-www.stanford.edu/Saccharomyces/ ). The resulting peptide matches were scored by SEQUEST, and protein identifications were considered valid if the identified protein contained at least two peptides with Xcorr scores above 2.0.

Glutathione S-transferase (GST) pull-down assays.Yeast strains SY4050 and SY3972 were grown at 30°C in 50 ml of YP containing 2% raffinose to an optical density at 600 nm of 0.75. Expression of P_GAL1-GST-HisX6-AHP1 and P_GAL1-GST-HisX6 was induced by the addition of 2% galactose, and the cells were grown for an additional 4 h at 30°C. The cells were harvested by centrifugation, converted to spheroplasts as described above, and lysed in 500 μl of buffer A. The lysates were clarified by centrifugation at 12,000 × g and bound in batch for 1 h to 100 μl of glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech, Piscataway, N.J.). The beads were collected by centrifugation at 500 × g for 5 min, washed four times in phosphate-buffered saline, eluted in loading buffer, and applied to a 10% acrylamide gel. Following electrophoresis, protein bands were detected by Western blot analysis using a previously described purified anti-Urm1p antibody (17).

Invasive-growth assays.For the plate-washing assay, strains were patched on YPD plates and incubated at 30°C. After 2 days of incubation, the plates were washed under a stream of running water as the surface was gently rubbed with a finger to remove cells not in the agar, and invasive growth was scored (45).

Urm1p is conjugated to Ahp1p.In order to confirm that Urm1p was interacting with Ahp1p, we used a GST-tagged version of Ahp1p. Purification of Ahp1p from wild-type extracts and immunoblotting with Urm1p-specific antibodies indicated that a 32-kDa species of Ahp1p contained Urm1p (Fig. 2, right lane). These bands were not detected in extracts from cells expressing GST alone (Fig. 2, left lane). Intriguingly, either Urm1p itself or Ahp1p may undergo posttranslational modification, since the conjugated product appears as a doublet.

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FIG. 2.

Urm1p coimmunoprecipitates with Ahp1p from whole-cell extracts. Exponential cultures of haploid strains SY4050 (wild type) carrying GST-HisX6 and SY3972 (wild type) carrying GST-HisX6-Ahp1p were lysed in the presence of 20 mM N-ethylmaleimide. The lysates were analyzed by SDS-PAGE and immunoblot analysis using affinity-purified polyclonal anti-Urm1p. The bands corresponding to Urm1p conjugated to Ahp1p are indicated.

To further support the idea that Urm1p was conjugated to Ahp1p, we performed Western analysis using the anti-Urm1p antibody on strains lacking URM1, UBA4, and AHP1. Loss of AHP1 resulted in the disappearance of the ∼32-kDa protein, but did not affect the abundance of the ∼75-, 80-, 89-, and 125-kDa polypeptides (Fig. 3, lane ahp1Δ). However, loss of URM1 and UBA4 eliminated the ∼32-kDa protein, as well as many other polypeptides of higher molecular mass (Fig. 3, lanes urm1Δ and uba4Δ). Collectively, these results support the conclusion that Ahp1p is one of the several targets of the urmylation pathway.

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FIG. 3.

A 32-kDa band disappears in ahp1Δ cells. Strains SY3358 (wild-type), SY3806 (urm1Δ), SY3805 (uba4Δ), and SY3815 (ahp1Δ) were grown to mid-log phase in YEPD at 30°C. The lysates were analyzed by SDS-PAGE and immunoblot analysis using affinity-purified polyclonal anti-Urm1p antibodies and monoclonal antibodies to Dpm1p (to confirm equal protein loading). The L bracket designates high-molecular-weight Urm1p-modified species, while the asterisk designates the position of a 32-kDa Urm1p-modified species.

Ahp1p is not involved in budding or invasion.Previously, potential functions for the urmylation pathway in budding and nutrient sensing were identified. It was shown that the simultaneous loss of Urm1p and Cla4p, a p21-activated kinase that functions in budding, is lethal (16). The simultaneous loss of Cla4p and urmylation pathway components caused cells to arrest with a severe defect in budding, suggesting that the urmylation pathway has a role in vegetative growth. To begin to investigate the function of Urm1p conjugation to Ahp1p, we tested whether the loss of Ahp1p was essential in a cla4Δ background. Unlike uba4 and urm1 mutations, mutations in ahp1 were not synthetically lethal with cla4Δ (data not shown).

The other role identified for the urmylation pathway is related to nutrient sensing. When haploid cells are starved for nutrients, the cells become elongated, bud primarily from one pole, and can invade the agar substratum. Previously, we showed that the urmylation pathway has a role in invasive growth (17). In order to determine if Ahp1p is the target of the urmylation pathway during invasion, we tested whether the loss of Ahp1p causes a defect in invasion. While deletion of UBA4 and URM1 rendered cells unable to invade agar under starvation conditions (data not shown), deletion of AHP1 did not affect invasive growth. Therefore, Ahp1p does not appear to play a role in invasion.

Potential role for Ahp1p in TOR signaling.Loss of the urmylation pathway also resulted in hypersensitivity to rapamycin (a macrocyclic antibiotic) (17). Rapamycin inhibits Tor1p and Tor2p, ultimately resulting in cellular responses characteristic of nutrient deprivation through a mechanism involving translational and transcriptional arrest (3, 19, 27, 30). Since Ahp1p appears to be a target of urmylation, we tested whether ahp1 mutants are hypersensitive to rapamycin and if the mutations exhibited genetic interaction with TOR pathway mutations. A gln3Δ mutant, which is defective for a GATA-type transcription factor regulated by the TOR kinases and by the Ure2p repressor (5, 7, 44), served as a rapamycin-resistant control strain. A ure2Δ mutant served as a control for rapamycin sensitivity. We found that ahp1 null mutants were unable to grow after 5 days on medium containing rapamycin (data not shown). Moreover, even if the ahp1 null mutants were carrying a wild-type copy of TOR2 on a plasmid, the cells were still unable to grow after 5 days on medium containing rapamycin (Fig. 4A). The sensitivity to rapamycin was not due to a reduction in protein synthesis or a general drug sensitivity, because the ahp1 null mutants were not sensitive to cycloheximide (data not shown). To verify that the sensitivity of these mutants to rapamycin reflected inactivity of the TOR pathway, we measured the rapamycin sensitivities of ahp1Δ mutants carrying a plasmid-borne TOR2S1972I (TOR2-1) allele (35). This allele of TOR2 has previously been shown to confer rapamycin resistance on many sensitive mutants. We found that all the double mutants were no longer sensitive to rapamycin (Fig. 4B). In contrast, the ahp1Δ mutants carrying a wild-type version of TOR2 on a plasmid were still sensitive to rapamycin (data not shown). These results suggest that, like the urmylation pathway mutants, ahp1 mutants are hypersensitive to rapamycin because of inhibition of TOR pathway signaling.

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FIG. 4.

Ahp1p functions in TOR signaling. (A) Strains SY3836 (gln3Δ), BY4741 (wild type [WT]), SY3839 (urm1Δ), SY3840 (uba4Δ), SY3845 (ure2Δ), and SY4121 (ahp1Δ) carrying pTOR2 (pML40) were grown to mid-log phase in YEPD at 30°C. A serial dilution (1/10) was performed starting with 10,000 cells. Cells were spotted onto either SD-LEU (left) or SD-LEU plus 25 nM rapamycin (right) and grown for 3 days at 30°C. (B) Strains SY3836 (gln3Δ), BY4741 (wild-type), SY3839 (urm1Δ), SY3840 (uba4Δ), SY3845 (ure2Δ), and SY4121 (ahp1Δ) carrying pTOR2-1 (pML41) were grown to mid-log phase in YEPD at 30°C. A serial dilution (1/10) was performed starting with 10,000 cells. Cells were spotted onto SD-LEU (left) or SD-LEU plus 25 nM rapamycin (right) and grown for 3 days at 30°C.

Urmylation is involved in oxidative stress.Ahp1p is believed to function as an antioxidant specific for t-BOOH and the thiol oxidant diamide (24, 32, 42). We therefore tested whether the loss of the urmylation pathway results in sensitivity to either t-BOOH or diamide. The urm1 and the uba4 mutant strains were sensitive to diamide (Fig. 5B). In fact, the loss of URM1 or UBA4 caused cells to be more sensitive to diamide than did loss of AHP1. In contrast, although the ahp1 null mutant strain was hypersensitive to t-BOOH compared to the wild-type control, the urm1 and the uba4 mutant strains were not sensitive to t-BOOH (Fig. 5A). Taken together, these results suggest that the urmylation pathway has an antioxidant role specifically toward diamide in vivo. Furthermore, these results also suggest that the conjugation of Urm1p to Ahp1p affects the antioxidant role of Ahp1p toward diamide but not toward t-BOOH.

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FIG. 5.

Sensitivities of the urmylation pathway mutants to oxidative stress. (A) Loss of the urmylation pathway does not result in sensitivity to t-BOOH. Strains BY4741 (wild type [WT]), SY4119 (ahp1Δ), SY3839 (urm1Δ), SY3840 (uba4Δ), SY4122 (ahp1Δ urm1Δ), SY4123 (ahp1Δ uba4Δ), and SY4124 (urm1Δ uba4Δ) were grown to mid-log phase in YEPD at 30°C. A serial dilution (1/5) was performed starting with 10,000 cells. Cells were spotted onto either YEPD plus 0.4 mM t-BOOH (left), YEPD plus 1 mM t-BOOH (middle), or 1.5 mM t-BOOH (right) and grown for 3 days at 30°C. (B) Loss of Ahp1p and the urmylation pathway results in diamide sensitivity. Strains BY4741 (wild type), SY4119 (ahp1Δ), SY3839 (urm1Δ), SY3840 (uba4Δ), SY4122 (ahp1Δ urm1Δ), SY4123 (ahp1Δ uba4Δ), and SY4124 (urm1IΔ uba4IΔ) were grown to mid-log phase in YEPD at 30°C. A serial dilution (1/5) was performed starting with 10,000 cells. Cells were spotted onto either YEPD (left), YEPD plus 1 mM diamide (middle), or 1.3 mM diamide (right) and grown for 3 days at 30°C.

Given that Ahp1p is urmylated and controls sensitivity to t-BOOH and to diamide, we asked whether the state of Ahp1p urmylation was modulated by treatment with either of these agents. We observed a modest increase in the Ahp1p-Urm1p conjugate when cells were treated with diamide (Fig. 6). In contrast, the Ahp1p-Urm1p conjugate was absent from cells treated with t-BOOH. Whether urmylation of Ahp1p is blocked by t-BOOH or Ahp1p is degraded under these conditions cannot be determined from these data, though the former possibility seems more likely. Nonetheless, the salient point is that urmylation of Ahp1p is differentially affected by diamide and t-BOOH, concordant with the observation that the loss of urmylation causes a differential effect on sensitivity to these agents.

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FIG. 6.

The Ahp1p-Urm1p conjugate is affected by diamide and t-BOOH treatment. Strain SY3357 (wild type) was grown to mid-log phase in YEPD at 30°C. Cells were then grown in YEPD at 30°C in the presence of 1.5 mM diamide or 1.3 mM t-BOOH or without drug treatment for 4 h. The lysates were analyzed by SDS-PAGE and immunoblot analysis using affinity-purified polyclonal anti-Urm1p antibodies and monoclonal antibodies to Dpm1p.

Conjugation of Urm1p to Ahp1p.Only a single copy of Urm1p appears to be attached to each Ahp1p polypeptide. Two species of urmylated GST-HisX6-Ahp1p were detected by Western blotting. One is the expected size for urmylated GST-HisX6-Ahp1p. The second band is not sufficiently larger to contain a second Urm1p moeity. This result suggests that either Ahp1p or Urm1p is posttranslationally modified (Fig. 2). Since our previous results suggest that there is a higher-molecular-weight Urm1p species, it is possible that Urm1p is the protein that is modified (17).

Although we have not identified which of the 14 lysines on Ahp1p is modified by Urm1p, two results support the idea that Ahp1p is a target and not an enzyme of the urmylation pathway. First, although the Ahp1p-Urm1p conjugate is detected only when 20 mM N-ethylmaleimide is present during cell lysis, these samples were resuspended in loading buffer that contained 2-mercaptoethanol (βME) (17)(Fig. 3). Sample buffer containing this reducing agent should disrupt the Urm1 thiol esters of E1 or E2, whereas Urm1p-protein conjugates would be resistant to βME (21). Because the Urm1p-Ahp1p product is present when treated with βME, it seems likely that Ahp1p is a target of the urmylation pathway. Second, loss of AHP1 affects the presence of only the 32-kDa Urm1p-Ahp1p conjugate, not other urmylated targets (Fig. 3). If Ahp1p functioned as an E2 or an E3 for the urmylation pathway, then the loss of Ahp1p would be expected to affect at least some of these other bands. Together, our results suggest that Ahp1p is a target of the urmylation pathway.

Possible functions for Urm1p attachment to Ahp1p.When cells are exposed to elevated levels of reactive oxygen species, they suffer oxidative stress. Oxidative stress can lead to DNA damage, lipid peroxidation, and protein oxidation (22, 50). The altered diamide tolerance phenotype seen in the ahp1 null mutant is believed to be related to the generation of lipid hydroperoxides upon exposure to the oxidant diamide (32). Similarly, mutations in either URM1 or UBA4 cause cells to become hypersensitive to diamide. Our results therefore suggest a potential role for the urmylation pathway in an antioxidant activity specific for diamide. In keeping with this possibility, treatment of cells with diamide causes an increase, albeit a modest one, in the amount of urmylated Ahp1p. Interestingly, the loss of the urmylation pathway components causes cells to be more sensitive to diamide than does the loss of Ahp1p. One possible explanation for the difference in diamide sensitivity may be that Urm1p interacts with another antioxidant enzyme(s). In support of this possibility, previous results indicated that different peroxiredoxins in yeast might have redundant and nonredundant functions (42, 54).

We also identified a potential interaction between Ahp1p and the TOR pathway. As seen for loss of URM1 or UBA4, the loss of AHP1 confers sensitivity to rapamycin. Since loss of AHP1 confers rapamycin sensitivity, it might be an enhancer of TOR signaling or of a cellular process regulated by TOR. Recent results suggest that the TOR pathway may play a role in oxidative-stress response. In Schizosaccharomyces pombe, tor1+ is required for response to oxidative stress, as well as other stresses (53). Therefore, Urm1p targeting of Ahp1p may provide a link between the TOR pathway and oxidative-stress response in S. cerevisiae.

Implications of urmylation and oxidative-stress response in mammals.Damage to cells caused by oxidative stress has been implicated in aging (51), neurodegenerative diseases (6), diabetes (28), and cancer (2). In mammals, reactive oxygen species are a well-established signal for regulation of transcription factors, such as NF-κB (nuclear factor κB), Ap1 (activator protein 1), and p53 (tumor suppressor) (1, 13, 23, 38). In higher plants, as well as animals, reactive oxygen species are also established signals in response to wound healing (43, 46). Thus, a growing area of research is focused on trying to understand the mechanisms that protect cells from oxidative stress or promote cellular recovery. Potential homologues for Ahp1p have been identified in plants, animals, fungi, and prokaryotes (52). Sequence similarity with these other potential antioxidants is highest around a putative catalytic active site that may be important for the peroxidase function (cysteine 62 in Ahp1p) (52). Since there also appears to be a homologue to Urm1p in mammals, perhaps urmylation of Ahp1p is important in oxidative-stress response in mammals. Further characterization of Ahp1p and the attachment of Urm1p is necessary to determine if this conjugate is involved in an antioxidant mechanism and/or other functions in S. cerevisiae.