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Membrane and soluble substrates of the Doa10 ubiquitin ligase are degraded by distinct pathways

2006; Springer Nature; Volume: 25; Issue: 3 Linguagem: Inglês

10.1038/sj.emboj.7600946

1460-2075

Tommer Ravid, Stefan G. Kreft, Mark Hochstrasser,

Genetics and Neurodevelopmental Disorders

Article26 January 2006free access Membrane and soluble substrates of the Doa10 ubiquitin ligase are degraded by distinct pathways Tommer Ravid Tommer Ravid Search for more papers by this author Stefan G Kreft Stefan G Kreft Search for more papers by this author Mark Hochstrasser Corresponding Author Mark Hochstrasser Molecular Biophysics & Biochemistry, Yale University, New Haven, CT, USA Search for more papers by this author Tommer Ravid Tommer Ravid Search for more papers by this author Stefan G Kreft Stefan G Kreft Search for more papers by this author Mark Hochstrasser Corresponding Author Mark Hochstrasser Molecular Biophysics & Biochemistry, Yale University, New Haven, CT, USA Search for more papers by this author Author Information Tommer Ravid, Stefan G Kreft and Mark Hochstrasser 1 1Molecular Biophysics & Biochemistry, Yale University, New Haven, CT, USA *Corresponding author. Molecular Biophysics & Biochemistry, Yale University, 266 Whitney Avenue, New Haven, CT 06520, USA. Tel.: +1 203 432 5101; Fax: +1 203 432 5175; E-mail: [email protected] The EMBO Journal (2006)25:533-543https://doi.org/10.1038/sj.emboj.7600946 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The yeast Doa10 ubiquitin (Ub) ligase resides in the endoplasmic reticulum (ER)/nuclear envelope (NE), where it functions in ER-associated degradation (ERAD). Doa10 substrates include non-ER proteins such as the transcription factor Matα2. Here, we expand the range of Doa10 substrates to include a defective kinetochore component, a mutant NE membrane protein, and a substrate-regulated human ER enzyme. For all these substrates, Doa10 requires two Ub-conjugating enzymes, Ubc6 and Ubc7, as well as the Ubc7 cofactor Cue1. Based on a novel genomic screen of a comprehensive gene deletion library and other data, these four proteins appear to be the only nonessential and nonredundant factors generally required for Doa10-mediated ubiquitination. Notably, the Cdc48 ATPase facilitates degradation of membrane-embedded Doa10 substrates, but is not required for any tested soluble Doa10 substrates. This distinction is maintained even when comparing membrane and soluble proteins bearing the same degradation signal. Thus, while Doa10 ubiquitinates both membrane and soluble proteins, the mechanisms of subsequent proteasome targeting differ. Introduction Protein degradation plays an essential role in many biological processes. In eukaryotes, the most common mechanism for degrading intracellular proteins is by the ubiquitin (Ub)–proteasome system (Hochstrasser, 1996; Pickart, 2001; Varshavsky, 2005). Polymers of Ub are ligated to the substrate protein, leading to the recognition and destruction of the substrate by the 26S proteasome. For Ub–protein conjugation, Ub is first activated in an energy-dependent reaction by Ub-activating enzyme (E1), followed by transfer of the Ub to a Ub-conjugating enzyme (E2). The E2, together with a third factor, a Ub–protein ligase or E3, transfers Ub to the target protein. E3s are enzymes that stimulate E2-dependent ubiquitination of substrates; because they usually bind directly to substrates, E3s are often the principal factors dictating substrate recognition (Pickart, 2001). Substrates contain degradation signals or degrons that are recognized by an E3 or E3–E2 complex (Laney and Hochstrasser, 1999). Degrons vary greatly between substrates, and only a few have been characterized in detail. The Saccharomyces cerevisiae Matα2 transcription factor is a nuclear protein that is degraded by the Ub system. Two Ub pathways are required for normal rates of α2 degradation (Chen et al, 1993; Swanson et al, 2001). The first involves the E2s Ubc4 and Ubc5. The second pathway utilizes the endoplasmic reticulum (ER)-localized E2s Ubc6 and Ubc7 and the ER/nuclear envelope (NE) transmembrane E3 Doa10. The latter pathway recognizes a well-defined degron called Deg1 (Johnson et al, 1998). Whereas α2 degradation serves a regulatory purpose (Laney and Hochstrasser, 2003), many misfolded or otherwise faulty proteins are detected by ‘quality-control’ mechanisms and are also rapidly degraded by the Ub system. Failure of such quality control leads to the accumulation of misfolded proteins, which is associated with multiple human degenerative disorders (Ciechanover and Schwartz, 2004). A well-known site of protein quality control is the ER, where both luminal and membrane proteins, if not correctly folded or assembled, are degraded in a process called ER-associated degradation (ERAD). ERAD of membrane proteins involves their ubiquitination and retrotranslocation to the cytosol for degradation by the proteasome (Hampton, 2002; Hirsch et al, 2004). Unexpectedly, some of the same Ub enzymes that participate in the proteolysis of soluble cytosolic or nuclear proteins also mediate membrane protein degradation at the ER. Matα2 was the first identified substrate for Ubc6, Ubc7, and Doa10, but these enzymes also have membrane protein substrates. Two mutant plasma-membrane transporters, Pma1-D378N and Ste6–166, are rapidly degraded in the ER by the Doa10 pathway, as is Ubc6 itself (Swanson et al, 2001; Wang and Chang, 2003; Huyer et al, 2004; Vashist and Ng, 2004). The other major yeast ERAD pathway involves the Hrd1/Der3 E3 and Ubc7 E2, which also targets both naturally short-lived and aberrant proteins, the latter including a mutant vacuolar carboxypeptidase Y called CPY* (Hampton, 2002; Hirsch et al, 2004). However, one apparent difference between yeast Doa10 and Hrd1 is that the Doa10 pathway recognizes both membrane proteins and soluble proteins of the cytoplasm/nucleus, whereas Hrd1 recognizes only membrane or luminal substrates. The ability of the Doa10 pathway to recognize both membrane and nonmembrane proteins raises the question of whether both substrate classes have the same types of degrons. One difficulty in addressing this has been that all the known membrane substrates of Doa10 were aberrant proteins (except Ubc6, which is part of the Doa10 complex), while no soluble quality-control substrate had been identified. The recognition of regulatory and quality-control substrates might involve distinct features regardless of whether they are membrane or soluble proteins. To help untangle these factors, we sought and now report a number of new Doa10 targets, including a mutant nonmembrane protein subject to quality control and a functional membrane protein. Conversely, we have constructed a matched set of soluble and membrane proteins with the same degron and show that they are all efficient Doa10 substrates. We were also interested in whether the proteolytic pathways for soluble and membrane Doa10 substrates showed any divergence. Our data indicate that membrane-embedded Doa10 substrates, but not soluble ones, require the Cdc48-Ufd1-Npl4 ATPase complex and the Rad23 and Dsk2 poly-Ub receptors for their degradation. The distinction is maintained even if the membrane and soluble proteins carry the same degron. This is the first identified substrate property that predicts the proteasomal targeting route among different substrates sharing the same Ub ligase. For both classes of substrate, the same four proteins—Doa10, Ubc6, Ubc7, and Cue1—are necessary for ubiquitination. A novel genome-wide screen with a Deg1-based substrate as well as further analysis of a previous selection that used a mutant nuclear substrate (Kopski and Huffaker, 1997) both yielded only these factors. Our results indicate that a diverse array of substrates, both membrane and nonmembrane, can be recognized by an ER-embedded Doa10 complex, and they also suggest that naturally short-lived regulatory proteins and aberrant quality control targets are likely to share related recognition determinants. Results As a way to find new Doa10 substrates, we scanned the literature for proteins whose degradation in yeast appeared to depend strongly on both Ubc6 and Ubc7 because previously identified substrates of Doa10 required both of these E2s. Three candidate proteins were identified: mutant versions of yeast Ndc10 and Mps2, and the human type II iodothyronine deiodinase. A series of synthetic degron fusions were also regarded as potential targets. The Ndc10-2 kinetochore protein is a Doa10 pathway substrate One potential substrate was the mutant Ndc10-2 protein (Kopski and Huffaker, 1997). Ndc10 is a subunit of the centromeric DNA-binding CBF3 complex and also associates with intranuclear mitotic spindle microtubules (Muller-Reichert et al, 2003). The temperature sensitivity of the ndc10-2 mutant is suppressed by loss of Ubc6 or Ubc7 (Kopski and Huffaker, 1997). This might reflect metabolic stabilization of a partially functional Ndc10-2 protein. We combined the ndc10-2 allele with either cue1Δ (Cue1 anchors Ubc7 to the ER) or doa10Δ to determine if these Doa10 pathway components also suppressed ndc10-2 temperature sensitivity. This was indeed observed (not shown). To address whether suppression correlated with changes in the degradation of Ndc10-2, we analyzed its rate of disappearance after protein synthesis was blocked (Figure 1A). Minimal degradation of wild-type Ndc10 protein occurred during the 1 h chase, but mutant Ndc10-2 protein rapidly disappeared under these conditions. Most importantly, degradation of Ndc10-2 protein was strongly inhibited by the deletion of either CUE1 or DOA10. Figure 1.The Ndc10-2 kinetochore protein is a short-lived substrate of the Doa10 pathway. (A) Degradation of Ndc10-2 protein in doa10Δ and cue1Δ cells. Cycloheximide was added 15 min after shifting them to 37°C, and aliquots of cells were removed at the indicated times. Lysates were analyzed by anti-Ndc10 immunoblotting. A strain with the chromosomal NDC10 tagged with the TAP-tag coding sequence (lane 1) was used as a control for antibody specificity. Asterisk, a crossreacting protein that served as a loading control. (B) Two previously isolated suppressors of ndc10-2 are also defective for Deg1-mediated proteolysis. Strains carrying the suppressor mutations kis3–66 and kis4–14 were transformed with a LEU2 plasmid expressing the fusion Deg1-Ura3. Failure to degrade this protein rapidly allows growth on uracil. (C) Complementation analysis demonstrates that kis3–66 has a mutation in DOA10 and kis4–14 a mutation in CUE1. The strains on the upper half of the plate were mated to cue1Δ, while those on the bottom half were mated to doa10Δ cells. Two diploids from each cross were streaked on each plate. (D) Anti-Doa10 immunoblot analysis of kis mutants. Download figure Download PowerPoint Extragenic ndc10-2 suppressor mutations in DOA10 and CUE1 Kopski and Huffaker (1997) found four complementation groups in their screen for suppressors of ndc10-2. Two of them, kis1 and kis2, had mutations in UBC7 and UBC6, respectively (Table I). The genes affected by the kis3 and kis4 mutations were not identified. To test if kis3 and kis4 strains also harbored mutations in known or unknown components of the Doa10 pathway, we transformed them with a plasmid encoding Deg1-Ura3, a convenient reporter for Doa10 pathway function. Wild-type cells rapidly degrade the reporter protein. Ura3 is required for uracil synthesis, and its rapid degradation prevents cells from growing on medium lacking uracil (SD-ura) (Chen et al, 1993). Doa10 pathway mutants are strongly impaired in Deg1-Ura3 turnover, allowing growth on SD-ura. As shown in Figure 1B, the kis3 and kis4 mutants also grew well under these conditions. We next tested whether the kis3 and kis4 strains might have mutations in Cue1 or Doa10. The Deg1-Ura3-based growth assay was used for complementation analysis. Growth on SD-ura was observed only for the crosses between doa10Δ and kis3–66 and between cue1Δ and kis4–14 (Figure 1C). These data suggested that kis3–66 is a mutant allele of DOA10 and kis4–14 is a mutation in CUE1 (Table I). In support of this, no Doa10 protein was detected by anti-Doa10 immunoblotting in kis3–66 cells, unlike wild-type or kis4–14 cells (Figure 1D), and sequencing of the CUE1 gene from the kis4–14 strain revealed a single C-to-T transition at nucleotide 250 in the ORF, changing Q84 to a stop codon. Table 1. Gene assignments for the ndc10-2 suppressors (Kopski and Huffaker, 1997) Complementation group Alleles Gene Reference kis1 10 UBC7 Kopski and Huffaker (1997) kis2 3 UBC6 Kopski and Huffaker (1997) kis3 10 DOA10 This study kis4 1 CUE1 This study Synthetic degron-protein fusions are Doa10 substrates Another group of short-lived proteins that were identified as Ubc6/Ubc7/Cue1-dependent substrates were the artificial SL17/CL degron fusions of Gilon et al (2000). Short peptides derived from a library of random yeast DNA inserts fused downstream of either the Escherichia coli gene for β-galactosidase (βgal) or yeast URA3 conferred rapid degradation on the encoded proteins. Degradation of a βgal-SL17 fusion was strongly impaired in doa10Δ cells (Figure 2A). Consistent with this, when wild-type and doa10Δ cells expressing a Ura3-SL17 fusion were compared for growth on SD-ura, robust growth was only seen in doa10Δ (Figure 2B). Growth analysis of six different Ura3-CL degron fusions gave similar results. Interestingly, these fusions with Ura3 localized to the cytoplasm and were apparently excluded from the nucleus (Figure 2C), in contrast to Deg1-protein fusions, which concentrate in the nucleus. Figure 2.Doa10 substrates include a series of cytoplasmic synthetic degron fusions. (A) Degradation of a fusion of the SL17 degron with βgal measured by pulse-chase analysis at 30°C. (B) Growth on SD-ura of wild-type (WT) and doa10Δ (Δ) cells expressing fusions of the indicated degrons to Ura3. (C) Cytoplasmic localization of Ura3-HA-SL17 detected by anti-HA immunofluorescent staining in doa10Δ. Staining was similar in WT and doa10Δ cells, but much brighter in the latter; three fusions (SL17, CL1 and CL2) were tested and gave similar results. Scale bar, 5 μm. Download figure Download PowerPoint Diverse membrane protein targets of Doa10 The preceding data are consistent with the idea that Doa10 is principally involved in recognizing degradation determinants on the cytosolic side of the ER membrane, while Hrd1 may mostly monitor luminally exposed structural features (Vashist and Ng, 2004). Relatively few proteins have been analyzed, however, so it is not certain that this will be generally true. Specificity for the Hrd1 and Doa10 E3s might arise through their recognition of substrate features on the same side of the membrane or the partitioning of E3s and substrates to different membrane subdomains. A broader range of membrane substrates for both E3s will be needed to determine the relative importance of these different mechanisms. One candidate membrane substrate for Doa10 was the mutant Mps2-1 protein (McBratney and Winey, 2002). Mps2 is a single-pass membrane protein that helps tether the spindle pole body (SPB), the yeast equivalent of the vertebrate centrosome, to the NE. The thermolabile Mps2-1 point mutant is short-lived; when cells are shifted to high temperature, the protein disappears from SPBs (McBratney and Winey, 2002). The growth defect of mps2-1 cells at 36°C is suppressed by deleting CUE1, UBC6, or UBC7, but not HRD1, and the rapid degradation and SPB depletion of Mps2-1 protein is also strongly inhibited by cuelΔ. We deleted DOA10 from mps2-1 cells to determine if this would also suppress mps2-1 temperature sensitivity (Figure 3A). Four independent mps2-1 doa10Δ strains were tested. All showed strong suppression of the mps2-1 growth defect at 36°C, consistent with stabilization of a partially functional protein. Figure 3.Degradation of a mutant transmembrane NE protein and a human ER-localized enzyme requires Doa10. (A) Mps2-1 is degraded by the Doa10 pathway. The DOA10 locus was deleted in an mps2-1 strain; the double mutant is shown alongside the original mps2-1 mutant and a wild-type (WT) control. Cells were grown on YPD for 3 days. (B) Degradation of the human ER-localized Dio2 requires Doa10 in yeast. Anti-FLAG immunoblotting was carried out for the indicated strains expressing the deiodinase D2-FLAG from a galactose-regulated promoter. The two selenocysteine codons in Dio2 were changed to cysteine for expression in yeast (Botero et al, 2002). Last lane, WT strain transformed with empty vector. Download figure Download PowerPoint The human deiodinase D2 (Dio2) has its N-terminal end inserted in the ER membrane (Botero et al, 2002). This short-lived enzyme converts the thyroid hormone thyroxine (T4) to its active form; high levels of T4 increase the rate of Dio2 degradation. Dio2 proteolysis shows similar features when expressed in human and yeast cells. In particular, Ubc6 and Ubc7 have been implicated in Dio2 ubiquitination in both organisms, and T4 stimulates Dio2 degradation in yeast as well (Botero et al, 2002). We therefore asked whether Doa10 was important for Dio2 degradation (Figure 3B). As expected, deletion of either Ubc6 or Ubc7 caused much greater accumulation of Dio2 than in wild-type cells, where the protein was barely detectable. Similarly, high levels of Dio2 were observed in doa10Δ and doa10Δ hrd1Δ cells, but not in hrd1Δ. We conclude that the naturally short-lived Dio2 deiodinase is a substrate of Doa10 in yeast. Together with earlier data on membrane protein substrates of Doa10, the new findings with Mps2-1 and Dio2 indicate that many structurally diverse membrane proteins can be ubiquitinated by Doa10. Some of these can be considered quality-control substrates, whereas others are wild-type proteins that are rapidly degraded as part of their normal regulation. A genomic screen for genes required for Doa10 pathway function Besides E2 and E3 enzymes and the proteasome, additional proteins generally are required for efficient degradation of ERAD substrates. These proteins participate in various stages of the proteolytic pathway, such as substrate recognition, retrotranslocation, and trafficking to the proteasome (Hirsch et al, 2004). Previous genetic screens for factors involved in the degradation of α2 identified the E2 and E3 enzymes of the Doa10 pathway as well as proteasome subunits and proteasome regulators. Only the Ubc6 and Ubc7 E2s (and the Ubc7 cofactor Cue1) and the Doa10 E3 were shown to be crucial for Deg1-mediated ubiquitination. To identify potential additional factors that contributed to the ubiquitination and degradation of Deg1-bearing substrates, we performed a high-throughput screen of a library of ∼4800 viable yeast gene deletion strains using synthetic genetic array (SGA) technology. Our proteolytic reporter was a fusion of Deg1 to Ura3 and a triplicated HA epitope (Deg1-Ura3-3HA). Rapid degradation of the fusion protein in wild-type cells prevents them from forming colonies on SD-ura, but mutations that hinder its degradation lead to uracil prototrophy. The chromosomally integrated Deg1-URA3-3HA reporter was introduced into each of the single gene deletion yeast strains by the SGA method (Materials and methods). Over 99% of the 4753 deletion strains were successfully crossed and selected to carry both the reporter and the individual gene deletion. These were then pinned onto SD-ura plates. In all, 47 strains grew on the selective media. These strains, along with a negative control hrd1Δ strain, were transferred to a master plate and rescreened on SD-ura, leaving 17 mutants that consistently grew faster than controls (Table II; Supplementary Figure S1A). Four strains grew much faster than the rest: cue1Δ, doa10Δ, ubc7Δ, and nup120Δ. The first three inactivate known components of the Doa10 ubiquitination pathway (ubc6Δ was not in the library), while the fourth lacks the nuclear pore protein Nup120. Table 2. Mutants identified from genomic deletion screen with Deg1-Ura3-HA3 Systematic name Standard name RGa Functional description Ubiquitin conjugation YMR022W UBC7 4 Ubiquitin-conjugating enzyme YMR264W CUE1 4 Recruits Ubc7 to the ER YIL030C DOA10 4 E3 ubiquitin ligase Proteasome YDL020C RPN4 2 Transcriptional activator for proteasome genes YBR173C UMP1 3 20S proteasome maturation factor YDR363W-A SEM1 3 26S proteasome regulatory subunit Chromosome/cell cycle/DNA replication YGR188C BUB1 3 Protein kinase that functions in spindle checkpoint YOR026W BUB3 3 Spindle checkpoint protein; binds Bub1 YPL008W CHL1 2 Helicase, required for sister chromatid cohesion YPR141C KAR3 3 Kinesin related, required for sister chromatid cohesion YHR191C CTF8 2 Replication factor, required for sister chromatid cohesion Telomerase YLR233C EST1 2 Telomerase holoenzyme subunit YLR318W EST2 2 Telomerase catalytic subunit YIL009C-A EST3 2 Telomerase holoenzyme subunit Other YPR131C NAT3 3 Catalyzes acetylation of N-terminal methionine of proteins YJL092W SRS2 3 DNA helicase, disassembles Rad51 filaments aRelative growth value was based on three to four independent growth assays (scales 1–4, 4=fastest). Among the deletion strains that grew on SD-ura plates but more slowly were strains lacking subunits or regulators of the proteasome: the Sem1 regulatory subunit, the Ump1 maturation factor, and the Rpn4 proteasomal gene regulator. Most proteasome components are essential for viability and were therefore not present in the deletion library. Deletions of two nonessential proteasome subunits, Pre9 and Rpn10, do not impair Deg1-mediated proteolysis (Velichutina et al, 2004; Verma et al, 2004), explaining why they were not isolated in our screen. Some strains showed very weak growth enhancements on SD-ura, but intriguingly, the mutations affected proteins from a very limited number of cellular regulatory systems (Table II). Three telomerase subunits were identified, as were several proteins linked to the mitotic checkpoint and chromatid cohesion. Loss of the Nat3 Nα-acetyltransferase also enhanced growth. These findings suggest that these factors might modulate Doa10 pathway function, possibly indirectly. For representative candidate strains, degradation of Ubc6-HA and Deg1-βgal test substrates was monitored (Supplementary Figure S1B). As predicted, degradation rates of the test substrates showed an inverse relationship with colony growth on SD-ura. Further analysis of the nup120Δ strain revealed that an unlinked lesion was responsible for the degradation defect (not shown). Based on complementation analysis and anti-Doa10 immunoblotting, the strain had a cryptic mutation in DOA10 (Supplementary Figure S1C). In summary, a comprehensive screen of the nonessential genes in S. cerevisiae has identified a very small number that are required for Deg1-mediated degradation, and these are precisely the same genes previously shown to encode the Ub-ligation enzymes of the Doa10 pathway. Not insignificantly, the identical set of genes was identified in the selection for ndc10-2 suppressors (Table I). The above analyses suggest that Doa10 pathway substrates require a common set of factors, including Doa10, Ubc6, Ubc7, and Cue1. Surprisingly, Cue1 was reported to have a much more limited role than Ubc6 or Ubc7 in degrading α2 itself (Lenk and Sommer, 2000). Cue1 is a transmembrane ER receptor for Ubc7. We re-examined the contribution of Cue1 to α2 degradation by deleting CUE1 or both CUE1 and UBC4 (Supplementary Figure S2). The half-life of α2 increased ∼2-fold in cue1Δ cells, similar to what was seen in ubc7Δ (Chen et al, 1993). Moreover, a synergistic impairment of degradation was observed in cue1Δ ubc4Δ cells, as expected, if both major α2 ubiquitination pathways were blocked. The ∼8-fold inhibition was similar to that previously observed in ubc4Δ ubc7Δ mutants. We conclude that Cue1 is required in the Doa10 pathway of α2 ubiquitination. Membrane and soluble Doa10 substrates differ in their Cdc48 dependence All of these genetic analyses suggested that Doa10, Ubc6, Ubc7, and Cue1 were the only nonessential and nonredundant factors encoded in the yeast genome that were required for efficient Doa10-dependent protein ubiquitination. However, it was possible that there were essential (or redundant) proteins—other than the proteasome—that also contributed to Doa10-dependent proteolysis. We examined the role of one such essential protein complex directly and also asked whether different classes of substrates shared all steps between Ub addition and proteolysis by the proteasome. Different ubiquitinated proteins have distinct adaptors that bind to polyubiquitinated substrates and may ferry them to the 26S proteasome (Kim et al, 2004; Verma et al, 2004). Membrane substrates ubiquitinated by Hrd1 or Doa10 need the three-component Cdc48-Npl4-Ufd1 ATPase complex for their degradation (Bays et al, 2001; Wang and Chang, 2003; Huyer et al, 2004). The Cdc48 AAA ATPase can help extract proteins from the membrane (Ye et al, 2001), but the complex, which binds both poly-Ub and the proteasome, might also act more generally as a proteasome adaptor (Dai et al, 1998). Consistent with this latter idea, Cdc48 is also required for degradation of some nonmembrane proteins (Johnson et al, 1995; Cao et al, 2003). To test the involvement of the Cdc48 complex in the degradation of nonmembrane Doa10 substrates, we measured the turnover of α2 and a series of short-lived Deg1 fusion proteins in various mutants. As expected, in cells lacking the Doa10 pathway (ubc6Δ), α2 was stabilized ∼2–3-fold relative to wild-type cells (Figure 4A). In contrast, mutations in CDC48, NPL4, or UFD1 had no detectable effect on its degradation. Similarly, mutations in these same Cdc48 complex components failed to alter the degradation of either Deg1-βgal or Deg1-Ura3 (Figure 4B and not shown). Simultaneous measurement of Deg1-βgal and CPY*-HA degradation in cdc48 cells showed that the latter was stabilized, as expected (Figure 4C). Figure 4.The Cdc48 ATPase complex is required for Doa10 membrane substrates, but not α2 and soluble Deg1-bearing substrates. (A) Pulse-chase analysis of α2 degradation in Cdc48 complex mutants. Proteins were precipitated with anti-α2 antibodies. (B) Loss of Deg1-βgal is not affected in mutants lacking specific Cdc48 complex components. Loss of βgal activity was followed after the addition of cycloheximide. Three transformants of each strain were assayed. Error bars are obscured by symbols at some data points. (C) Degradation of Deg1-βgal is not impaired in cdc48-3 cells, in contrast to that of CPY*-HA. Cells cotransformed with plasmids encoding the respective proteins were assayed by cycloheximide chase and immunoblotting with antibodies to βgal and HA. (D) Degradation of the Ubc6-HA membrane protein requires the Cdc48 complex. Degradation was assayed by cycloheximide chase and anti-HA immunoblotting. (E) Degradation of Ubc6-HA, but not Deg1-βgal, requires Ubx2, an ER receptor for Cdc48. Protein turnover was assayed as in (C). (F) Degradation of Deg1-βgal and Ubc6-HA in ufd1-2 cells. Strains cotransformed with expression plasmids for the two proteins were assayed as in (C). (G) Rad23 and Dsk2 contribute to Ubc6-HA, but not Deg1-βgal degradation. Strains cotransformed with expression plasmids for the two proteins were assayed at 30°C as in (C). Download figure Download PowerPoint We also tested the degradation of fusions of the Deg1 degron to a single GFP, Deg1-GFP (Bays et al, 2001), or a tandem pair of GFPs, Deg1-GFP2 (Lenk and Sommer, 2000). Both proteins still required the Doa10 pathway for maximal degradation, although this dependence was notably weaker than for the above Deg1 reporters, presumably because an additional degradation pathway(s) can recognize the GFP constructs (Supplementary Figure S3). Although we consider these less than ideal test substrates, neither Deg1-GFP nor Deg1-GFP2 was degraded more slowly in any tested mutant of the Cdc48 complex. Others had previously tested the same GFP fusions in Cdc48 complex mutants. Our data agree with those of Bays et al (2001) and Medicherla et al (2004), but not Verma et al (2004) or Neuber et al (2005). The reason for the discrepancies is unknown, but might reflect effects on Doa10-independent pathways acting on these fusions. Until recently, mutant versions of the Pma1 and Ste6 transporters were the only Doa10 membrane substrates tested for Cdc48-dependent degradation (Wang and Chang, 2003; Huyer et al, 2004), so it was possible that their Cdc48 dependence was unusual, perhaps reflecting their large size or large number of TM segments. We therefore tested Ubc6, a small single TM substrate of Doa10. Unlike the different soluble substrates we tested, this short-lived enzyme was strongly stabilized in the cdc48, npl4, ufd1, and ubx2 mutants (Figure 4E and F). This is consistent with a very recent report on the same substrate (Neuber et al, 2005). Ubx2, an ER membrane protein, helps dock Cdc48 to the ER (Schuberth and Buchberger, 2005; Neuber et al, 2005). Turnover of Deg1-βgal in cells lacking Ubx2 was not impaired (Figure 4E). Notably, Ubc6-HA was stabilized in ufd1-2 cells, whereas Deg1-βgal coexpressed in the same cells was unaffected (Figure 4F). Finally, loss of the Rad23 and Dsk2 proteins, which function as (partially redundant) adaptors between polyubiquitinated substrates and the proteasome, also specifically stabilized Ubc6-HA, but not coexpressed soluble Deg1-βgal (Figure 4G). From the above data and from published results with mutant membrane transporters, it appears that the Cdc48 ATPase complex and Rad23/Dsk2 are crucial for the degradation of membrane