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. 2005 Jan;25(1):403-13.
doi: 10.1128/MCB.25.1.403-413.2005.

Proteasome-mediated Degradation of Cotranslationally Damaged Proteins Involves Translation Elongation Factor 1A

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Proteasome-mediated Degradation of Cotranslationally Damaged Proteins Involves Translation Elongation Factor 1A

Show-Mei Chuang et al. Mol Cell Biol. .
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Abstract

Rad23 and Rpn10 play synergistic roles in the recognition of ubiquitinated proteins by the proteasome, and loss of both proteins causes growth and proteolytic defects. However, the physiological targets of Rad23 and Rpn10 have not been well defined. We report that rad23Delta rpn10Delta is unable to grow in the presence of translation inhibitors, and this sensitivity was suppressed by translation elongation factor 1A (eEF1A). This discovery suggested that Rad23 and Rpn10 perform a role in translation quality control. Certain inhibitors increase translation errors during protein synthesis and cause the release of truncated polypeptide chains. This effect can also be mimicked by ATP depletion. We determined that eEF1A interacted with ubiquitinated proteins and the proteasome following ATP depletion. eEF1A interacted with the proteasome subunit Rpt1, and the turnover of nascent damaged proteins was deficient in rpt1. An eEF1A mutant (eEF1A(D156N)) that conferred hyperresistance to translation inhibitors was much more effective at eliminating damaged proteins and was detected in proteasomes in untreated cells. We propose that eEF1A is well suited to detect and promote degradation of damaged proteins because of its central role in translation elongation. Our findings provide a mechanistic foundation for defining how cellular proteins are degraded cotranslationally.

Figures

FIG. 1.
FIG. 1.
eEF1A can suppress the sensitivity of rad23Δ rpn10Δ to protein synthesis inhibitors. (A) eEF1A (TEF1) was expressed in rad23Δ rpn10Δ on low-copy-number (CEN) and high-copy-number (2μm) plasmids. Ten-fold serial dilutions were spotted onto agar medium and incubated at the permissive (24°C) and semipermissive (30°C) temperatures. (B) Wild-type and rad23Δ rpn10Δ mutant strains were transformed with a control 2μm plasmid, or the same plasmid expressing eEF1A, and spotted on medium containing cycloheximide (0.001 mM), hygromycin B (0.2 mM), or paromomycin (1 mM). WT, wild type.
FIG. 2.
FIG. 2.
eEF1A interacts with the 26S proteasome. (A) Pre1-FLAG was expressed in wild-type (WT) (lanes 2 and 5) and rad23Δ cells (lanes 3 and 6). Wild-type cells lacking Pre1-FLAG were also examined (lanes 1 and 4). Pre1-FLAG was immunoprecipitated (IP) and an immunoblot was probed with antibodies against eEF1A and Rpt1. (B) eEF1A was immunoprecipitated from extracts described in panel A, and an immunoblot was incubated with anti-FLAG antibody. (C) A yeast strain expressing epitope-tagged 20S subunit (Pup1-HA) and 19S subunit (Rpn8-V5) were grown to exponential phase and extracts incubated with V5 antibodies. An immunoblot was sequentially incubated with antibodies against eEF1A and HA. (D) Total cell extracts (2 mg) were resolved in Superose 6, and aliquots were examined by immunoblotting. The sedimentation positions of eEF1A, Pre1-FLAG, and Rpt1 were determined by immunoblotting. (E) Pre1-FLAG was immunoprecipitated from yeast cells expressing wild-type or mutant eEF1A proteins. The coprecipitation of eEF1A and Rpt1 with Pre1-FLAG was determined.
FIG. 3.
FIG. 3.
eEF1A interacts with proteasome subunit Rpt1. (A) Pre1-FLAG was purified from wild-type (WT; lanes 1, 3, 6, and 8), rpn2 (lanes 2 and 7), rpt1 (lanes 4 and 9), and rpt6 (lanes 5 and 10), and the copurification of eEF1A with the proteasome was determined. The strain background for rpn2 differed from that harboring rpt6 and rpt1 mutations. (B) rpt1 was grown at 24, 30, and 37°C, and Pre1-FLAG was precipitated. The levels of eEF1A and Rpt1 were examined. Stability of the proteasome at high temperature (37°C) was unaffected, as determined by the constant levels of Rpt1 that was precipitated from the wild-type and rpt1 strains. (C) Rpt1 was isolated from Escherichia coli and 100 ng of the purified protein was resolved in the right lane. Rpt1 was applied to immobilized GST-eEF1A, GST-Rpn8, GST-S5a, and GST, and >90% of the input Rpt1 bound GST-eEF1A. (D) The positions of purified Rpt1 (panel 3) and GST-eEF1A (panel 2) in Superose 6 are compared to bovine serum albumin (panel 1). Ten-fold less Rpt1 and GST-eEF1A were combined (to minimize aggregation or nonspecific binding), incubated at 4°C for 2 h, and resolved in Superose 6 (panels 4 and 5). Fractions were incubated with glutathione-Sepharose (GST pulldown [GST-PD]) and Rpt1 was coprecipitated with GST-eEF1A (panels 6 and 7). Dashed lines indicate the position of free eEF1A. IP, immunoprecipitation.
FIG. 4.
FIG. 4.
eEF1A interacts with ubiquitinated proteins, following ATP depletion. (A) GST and GST-UBA1 were expressed in wild-type cells that were either untreated (−) or subjected to ATP-depleting conditions (+). Protein extracts were incubated with glutathione-Sepharose and precipitated material was resolved by SDS-PAGE and transferred to a nitrocellulose filter. The interaction between GST-UBA1 and high-molecular-weight Ub cross-reacting material [Ub(n)] was confirmed in both extracts (lanes 3 and 4; arrow). Ubiquitinated proteins were not recovered with GST (lanes 1 and 2). (B) eEF1A was purified with GST-UBA1 (lane 8) only from ATP-depleted extracts (lane 8), although it was expressed at equivalent levels in both conditions (lanes 1 to 4). (C) GST-UBA1 was purified from untreated and ATP-depleted extracts, and the copurification of eEF1A and Pre1-FLAG was determined over time. Increasing amounts of eEF1A were bound to ubiquitinated proteins following ATP depletion, while progressively lower levels of Pre1-FLAG were detected with GST-UBA1. Control (Unt; time 0 and time 120 min) samples are present in lanes 1 and 8. Similarly, Pre1-FLAG was purified from the same extracts and the rapid association of eEF1A with the proteasome was confirmed. A detectable loss of Rpt1 over time was observed, consistent with slow dissociation of the 19S and 20S particles. (D) The increased association between eEF1A and the proteasome is also observed following treatment of cells with canavanine (100 μg/ml), neomycin (2 mM), and paromomycin (1 mM). The increased eEF1A-proteasome binding after 2 h was ∼8-fold in the presence of canavanine. Approximately twofold increased binding was observed in the presence of neomycin and paromomycin, respectively. (E) The interaction between eEF1A and various subfragments of Rad23 was examined in the absence (−) and presence (+) of ATP depletion. A strong interaction was detected with intact Rad23 (GST-Rad23; lane 4) and with GST-UBA1 (lane 6). In contrast, eEF1A was not recovered with GST or GST-UBA2, and very low levels were detected with GST-UbL. (F) Purified His6-eEF1A (100 ng each in lanes 4 to 6) was incubated with 50 ng tetra-Ub, and eEF1A was immunoprecipitated using anti-His6 antibodies. Although eEF1A was efficiently purified, tetra-Ub was not detected. Lanes 1 and 2 contained 10 and 50 ng of tetra-Ub. Lane 3 did not contain His6-eEF1A. In lanes 4 to 6, the incubation was performed in the presence of 50, 150, and 250 mM NaCl. IP, immunoprecipitation.
FIG. 5.
FIG. 5.
eEF1A can bind a normally stable protein in the presence of translation damage. (A) A yeast strain expressing Pre1-FLAG and Met-βgal was incubated with 2,4-DNP plus 2-DG, and culture samples were examined at the indicated times. Protein extract (50 μg) was separated by SDS-PAGE, transferred to nitrocellulose, and incubated with antibodies against βgal. Lane 1 is an untreated sample. Even-numbered lanes represent the untreated samples, while odd-numbered lanes show the progressive inhibition of protein synthesis following ATP depletion. (The same protein extracts were examined in panels A to D, and the lane numbers indicated at the bottom of panel D correspond to all the panels.) (B) Pre1-FLAG was immunoprecipitated (IP) and the rapid copurification of eEF1A was confirmed (see odd lanes). However, eEF1A abundance in the extracts was unaffected (Extract). (C) The filter examined in panel B above was incubated with antibodies against Ub, and high-molecular-weight Ub cross-reacting material was detected in association with the proteasome only in ATP-depleted extracts [upper panel; arrow, Ub(n)]. Equal amount of protein extracts were also resolved and incubated with antibodies against Ub (lower panel). (D) The filter shown in panel B above was incubated with antibodies against Met-βgal, and increasing levels of full-length and truncated fragments were detected (arrow). (E) A direct interaction between eEF1A and Met-βgal, following ATP depletion, was confirmed by immunoprecipitating Met-βgal and reacting an immunoblot with antibodies against eEF1A. As noted above, the rapid interaction occurred primarily in ATP-depleted samples.
FIG. 6.
FIG. 6.
rpt1 is defective in degrading nascent proteins. (A) Wild-type and rpt1 strains were subjected to ATP depletion conditions. In Fig. 5, yeast cells were grown at 30°C, while in the experiments described here, yeast cells were grown at the nonpermissive temperature for rpt1 (37°C for 6 h). Pre1-FLAG was precipitated and the levels of eEF1A in the proteasome were threefold lower in rpt1 than in the wild-type strain. (B) The same immunoblot was incubated with antibodies against βgal, and the level of Met-βgal in the proteasome was determined. (C) The levels of Met-βgal in cell extracts were determined. Note the much faster elimination of Met-βgal in this study (37°C) than in Fig. 5A (30°C). Essentially the same βgal cross-reacting bands were detected in the extract and in the proteasome (compare bands in panels B and C). (D) The levels of Ub cross-reacting material were investigated in wild-type and rpt1 proteasomes. Note that the film exposure for the wild-type samples (lanes 1 to 6) was 10 times longer than for the rpt1 samples (lanes 7 to 12). (E) The interaction between eEF1A and the proteasome was examined in other proteasome mutants (rpt4 and rpt6) following ATP depletion. WT, wild type; IP, immunoprecipitation.
FIG. 7.
FIG. 7.
(A) The growth of wild-type (WT) and tef1Δ tef2Δ expressing eEF1AD156N was determined in the presence of 2-μg/ml l-canavanine. Ten-fold dilutions were spotted, and growth was examined after 4 days. (B) GST-UBA1 was immunoprecipitated following ATP depletion, and the copurification of eEF1A and eEF1AD156N was determined. eEF1AD156N formed an efficient interaction with ubiquitinated proteins that were bound to the UBA1 domain of Rad23. A longer exposure (lower panel) revealed constitutive eEF1AD156N interaction with ubiquitinated proteins in untreated cells (lane 3). (C) The stability of a proteolytic substrate, Ub-Pro-βgal, was determined in cells expressing eEF1AD156N. Chase times are indicated (in minutes), and the stability (t1/2) was determined over the initial 30 min. Note that the initial incorporation of 35S label was similar in both strains (time 0). (D) The abundance of Met-βgal was examined in wild-type and tef1Δ tef2Δ expressing eEF1AD156N, in the presence and absence of 2-DG and 2,4-DNP. Total protein extracts were resolved by SDS-PAGE, transferred to nitrocellulose, and incubated with anti-βgal antibodies. High levels of Met-βgal were detected in the wild-type strain, while dramatically reduced levels were present in eEF1AD156N-expressing cells. In contrast, the levels of highly stable Pab1 were unchanged. The severely reduced level of Met-βgal is unlikely to be the result of a modest twofold reduction in translation rate in eEF1AD156N-expressing cells but is probably due to reduced fidelity and rapid cotranslational degradation. (E) We examined the stability of Ub-Pro-βgal, a well-characterized substrate of the Ub-proteasome, to determine if eEF1A suppressed the proteolytic defect of rad23Δ rpn10Δ. Yeast cells were labeled with [35S]Met plus [35S]Cys and chased in medium containing excess unlabeled amino acids and cycloheximide. Unlike the Rpn10 gene (which, as expected, was also isolated as a suppressor of rad23Δ rpn10Δ), we detected no significant recovery of degradation of Ub-Pro-βgal.
FIG. 8.
FIG. 8.
Model. The proposed model is consistent with published studies and the results described here. Nascent proteins that are misfolded are rapidly ubiquitinated and degraded by the proteasome. We suggest that eEF1A, Rad23, Rpn10, and the proteasome can affect cellular tolerance to translation inhibitors by regulating the degradation of nascent proteins. Because eEF1A binds ubiquitinated proteins and the proteasome after treatment with translational inhibitors, we propose that it can participate in the recognition and degradation of damaged nascent proteins.

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