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Functional Dissection of the Nascent Polypeptide-Associated Complex in Saccharomyces cerevisiae.

Ott AK, Locher L, Koch M, Deuerling E - PLoS ONE (2015)

Bottom Line: While loss of NAC does not cause phenotypic changes in yeast, the simultaneous deletion of genes coding for NAC and the chaperone Ssb (nacΔssbΔ) leads to strongly aggravated defects compared to cells lacking only Ssb, including impaired growth on plates containing L-canavanine or hygromycin B, aggregation of newly synthesized proteins and a reduced translational activity due to ribosome biogenesis defects.Expression of individual β-NAC, β'-NAC or α-NAC subunits as well as αβ'-NAC ameliorated protein aggregation in nacΔssbΔ cells to different extents while only β-NAC was able to restore growth defects suggesting chaperoning activities for β-NAC sufficient to decrease the sensitivity of nacΔssbΔ cells against L-canavanine or hygromycin B.Interestingly, deletion of the ubiquitin-associated (UBA)-domain of the α-NAC subunit strongly enhanced the aggregation preventing activity of αβ-NAC pointing to a negative regulatory role of this domain for the NAC chaperone activity in vivo.

View Article: PubMed Central - PubMed

Affiliation: Molecular Microbiology, Department of Biology, University of Konstanz, 78457, Konstanz, Germany.

ABSTRACT
Both the yeast nascent polypeptide-associated complex (NAC) and the Hsp40/70-based chaperone system RAC-Ssb are systems tethered to the ribosome to assist cotranslational processes such as folding of nascent polypeptides. While loss of NAC does not cause phenotypic changes in yeast, the simultaneous deletion of genes coding for NAC and the chaperone Ssb (nacΔssbΔ) leads to strongly aggravated defects compared to cells lacking only Ssb, including impaired growth on plates containing L-canavanine or hygromycin B, aggregation of newly synthesized proteins and a reduced translational activity due to ribosome biogenesis defects. In this study, we dissected the functional properties of the individual NAC-subunits (α-NAC, β-NAC and β'-NAC) and of different NAC heterodimers found in yeast (αβ-NAC and αβ'-NAC) by analyzing their capability to complement the pleiotropic phenotype of nacΔssbΔ cells. We show that the abundant heterodimer αβ-NAC but not its paralogue αβ'-NAC is able to suppress all phenotypic defects of nacΔssbΔ cells including global protein aggregation as well as translation and growth deficiencies. This suggests that αβ-NAC and αβ'-NAC are functionally distinct from each other. The function of αβ-NAC strictly depends on its ribosome association and on its high level of expression. Expression of individual β-NAC, β'-NAC or α-NAC subunits as well as αβ'-NAC ameliorated protein aggregation in nacΔssbΔ cells to different extents while only β-NAC was able to restore growth defects suggesting chaperoning activities for β-NAC sufficient to decrease the sensitivity of nacΔssbΔ cells against L-canavanine or hygromycin B. Interestingly, deletion of the ubiquitin-associated (UBA)-domain of the α-NAC subunit strongly enhanced the aggregation preventing activity of αβ-NAC pointing to a negative regulatory role of this domain for the NAC chaperone activity in vivo.

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Analysis of protein aggregation in nacΔssbΔ suppressed by NAC variants.a) 50 OD600 units of transformed yeast cells expressing the indicated NAC variants in the logarithmic phase were lysed and the aggregated protein material was isolated by sedimentation. Isolated aggregated fractions were separated by SDS-PAGE and visualized by Coomassie staining. b) Biological replicates of the experiment shown in a) for aggregated proteins of wt, ssbΔ and nacΔssbΔ cells (lanes 1–3), nacΔssbΔ + αβ-NAC (lane 8) and + αΔUBAβ-NAC (lane 10). The experiment was performed as in a). For better visualization the corresponding lanes were cut out from the same SDS-PAGE after Coomassie staining as indicated by black lines. c) Quantification of aggregated material using ImageJ shows the relative level of aggregated protein in relation to total protein amount, normalized to the mean value of wt replicates. Mean ± SD is shown from three experiments (n = 3).
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pone.0143457.g006: Analysis of protein aggregation in nacΔssbΔ suppressed by NAC variants.a) 50 OD600 units of transformed yeast cells expressing the indicated NAC variants in the logarithmic phase were lysed and the aggregated protein material was isolated by sedimentation. Isolated aggregated fractions were separated by SDS-PAGE and visualized by Coomassie staining. b) Biological replicates of the experiment shown in a) for aggregated proteins of wt, ssbΔ and nacΔssbΔ cells (lanes 1–3), nacΔssbΔ + αβ-NAC (lane 8) and + αΔUBAβ-NAC (lane 10). The experiment was performed as in a). For better visualization the corresponding lanes were cut out from the same SDS-PAGE after Coomassie staining as indicated by black lines. c) Quantification of aggregated material using ImageJ shows the relative level of aggregated protein in relation to total protein amount, normalized to the mean value of wt replicates. Mean ± SD is shown from three experiments (n = 3).

Mentions: The loss of NAC does not provoke protein aggregation while cells lacking the Ssb chaperone activity accumulate misfolded and insoluble proteins. However, defects in protein folding are much more pronounced in ssbΔ cells upon additional loss of NAC suggesting that these two ribosome-associated systems act in overlapping pathways to support the folding of newly synthesized proteins [18]. Therefore, we finally examined if NAC subunits expressed alone or in combination reduce the level of protein aggregation in nacΔssbΔ cells. Mutant cells were grown to exponential phase, harvested and after lysis the insoluble protein material was isolated by centrifugation (Fig 6). Equalized total lysates are shown in S2A Fig and served as a loading control. Three biologically independent experiments were conducted for each NAC variant to test the chaperone activity by analyzing the suppression of protein aggregation in nacΔssbΔ cells. The data were analyzed using one-way-between-groups ANOVA with post-hoc Tukey test [29] to assess significance. As shown previously, cells lacking Ssb and NAC revealed enhanced levels of insoluble protein material compared to ssbΔ cells (Fig 6A, lanes 1–3). We found that expression of αβ-NAC at wt levels significantly reduced protein aggregation in nacΔssbΔ cells (Fig 6A, lane 8; quantification in Fig 6C and S1 Table (p = 0)) suggesting that this heterodimer is a potent chaperone in vivo. The expression of the single NAC-subunits or of αβ’-NAC ameliorated protein aggregation as well, with some variances compared to the expression of αβ-NAC (Fig 6A, lanes 4, 7, 11 and S1 Table). This suggests that chaperone activity can be displayed by individual subunits and does not necessarily rely on the heterodimeric NAC complex. The chaperone activity of αβ-NAC and β-NAC critically depends on ribosome association as protein aggregation could not be prevented in nacΔssbΔ cells expressing the αβRRK/AAA-NAC or βRRK/AAA-NAC subunit (Fig 6A, lanes 6 and 9, Fig 6C, S1 Table (p = 0.24 and 0.921, respectively)).


Functional Dissection of the Nascent Polypeptide-Associated Complex in Saccharomyces cerevisiae.

Ott AK, Locher L, Koch M, Deuerling E - PLoS ONE (2015)

Analysis of protein aggregation in nacΔssbΔ suppressed by NAC variants.a) 50 OD600 units of transformed yeast cells expressing the indicated NAC variants in the logarithmic phase were lysed and the aggregated protein material was isolated by sedimentation. Isolated aggregated fractions were separated by SDS-PAGE and visualized by Coomassie staining. b) Biological replicates of the experiment shown in a) for aggregated proteins of wt, ssbΔ and nacΔssbΔ cells (lanes 1–3), nacΔssbΔ + αβ-NAC (lane 8) and + αΔUBAβ-NAC (lane 10). The experiment was performed as in a). For better visualization the corresponding lanes were cut out from the same SDS-PAGE after Coomassie staining as indicated by black lines. c) Quantification of aggregated material using ImageJ shows the relative level of aggregated protein in relation to total protein amount, normalized to the mean value of wt replicates. Mean ± SD is shown from three experiments (n = 3).
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Related In: Results  -  Collection

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pone.0143457.g006: Analysis of protein aggregation in nacΔssbΔ suppressed by NAC variants.a) 50 OD600 units of transformed yeast cells expressing the indicated NAC variants in the logarithmic phase were lysed and the aggregated protein material was isolated by sedimentation. Isolated aggregated fractions were separated by SDS-PAGE and visualized by Coomassie staining. b) Biological replicates of the experiment shown in a) for aggregated proteins of wt, ssbΔ and nacΔssbΔ cells (lanes 1–3), nacΔssbΔ + αβ-NAC (lane 8) and + αΔUBAβ-NAC (lane 10). The experiment was performed as in a). For better visualization the corresponding lanes were cut out from the same SDS-PAGE after Coomassie staining as indicated by black lines. c) Quantification of aggregated material using ImageJ shows the relative level of aggregated protein in relation to total protein amount, normalized to the mean value of wt replicates. Mean ± SD is shown from three experiments (n = 3).
Mentions: The loss of NAC does not provoke protein aggregation while cells lacking the Ssb chaperone activity accumulate misfolded and insoluble proteins. However, defects in protein folding are much more pronounced in ssbΔ cells upon additional loss of NAC suggesting that these two ribosome-associated systems act in overlapping pathways to support the folding of newly synthesized proteins [18]. Therefore, we finally examined if NAC subunits expressed alone or in combination reduce the level of protein aggregation in nacΔssbΔ cells. Mutant cells were grown to exponential phase, harvested and after lysis the insoluble protein material was isolated by centrifugation (Fig 6). Equalized total lysates are shown in S2A Fig and served as a loading control. Three biologically independent experiments were conducted for each NAC variant to test the chaperone activity by analyzing the suppression of protein aggregation in nacΔssbΔ cells. The data were analyzed using one-way-between-groups ANOVA with post-hoc Tukey test [29] to assess significance. As shown previously, cells lacking Ssb and NAC revealed enhanced levels of insoluble protein material compared to ssbΔ cells (Fig 6A, lanes 1–3). We found that expression of αβ-NAC at wt levels significantly reduced protein aggregation in nacΔssbΔ cells (Fig 6A, lane 8; quantification in Fig 6C and S1 Table (p = 0)) suggesting that this heterodimer is a potent chaperone in vivo. The expression of the single NAC-subunits or of αβ’-NAC ameliorated protein aggregation as well, with some variances compared to the expression of αβ-NAC (Fig 6A, lanes 4, 7, 11 and S1 Table). This suggests that chaperone activity can be displayed by individual subunits and does not necessarily rely on the heterodimeric NAC complex. The chaperone activity of αβ-NAC and β-NAC critically depends on ribosome association as protein aggregation could not be prevented in nacΔssbΔ cells expressing the αβRRK/AAA-NAC or βRRK/AAA-NAC subunit (Fig 6A, lanes 6 and 9, Fig 6C, S1 Table (p = 0.24 and 0.921, respectively)).

Bottom Line: While loss of NAC does not cause phenotypic changes in yeast, the simultaneous deletion of genes coding for NAC and the chaperone Ssb (nacΔssbΔ) leads to strongly aggravated defects compared to cells lacking only Ssb, including impaired growth on plates containing L-canavanine or hygromycin B, aggregation of newly synthesized proteins and a reduced translational activity due to ribosome biogenesis defects.Expression of individual β-NAC, β'-NAC or α-NAC subunits as well as αβ'-NAC ameliorated protein aggregation in nacΔssbΔ cells to different extents while only β-NAC was able to restore growth defects suggesting chaperoning activities for β-NAC sufficient to decrease the sensitivity of nacΔssbΔ cells against L-canavanine or hygromycin B.Interestingly, deletion of the ubiquitin-associated (UBA)-domain of the α-NAC subunit strongly enhanced the aggregation preventing activity of αβ-NAC pointing to a negative regulatory role of this domain for the NAC chaperone activity in vivo.

View Article: PubMed Central - PubMed

Affiliation: Molecular Microbiology, Department of Biology, University of Konstanz, 78457, Konstanz, Germany.

ABSTRACT
Both the yeast nascent polypeptide-associated complex (NAC) and the Hsp40/70-based chaperone system RAC-Ssb are systems tethered to the ribosome to assist cotranslational processes such as folding of nascent polypeptides. While loss of NAC does not cause phenotypic changes in yeast, the simultaneous deletion of genes coding for NAC and the chaperone Ssb (nacΔssbΔ) leads to strongly aggravated defects compared to cells lacking only Ssb, including impaired growth on plates containing L-canavanine or hygromycin B, aggregation of newly synthesized proteins and a reduced translational activity due to ribosome biogenesis defects. In this study, we dissected the functional properties of the individual NAC-subunits (α-NAC, β-NAC and β'-NAC) and of different NAC heterodimers found in yeast (αβ-NAC and αβ'-NAC) by analyzing their capability to complement the pleiotropic phenotype of nacΔssbΔ cells. We show that the abundant heterodimer αβ-NAC but not its paralogue αβ'-NAC is able to suppress all phenotypic defects of nacΔssbΔ cells including global protein aggregation as well as translation and growth deficiencies. This suggests that αβ-NAC and αβ'-NAC are functionally distinct from each other. The function of αβ-NAC strictly depends on its ribosome association and on its high level of expression. Expression of individual β-NAC, β'-NAC or α-NAC subunits as well as αβ'-NAC ameliorated protein aggregation in nacΔssbΔ cells to different extents while only β-NAC was able to restore growth defects suggesting chaperoning activities for β-NAC sufficient to decrease the sensitivity of nacΔssbΔ cells against L-canavanine or hygromycin B. Interestingly, deletion of the ubiquitin-associated (UBA)-domain of the α-NAC subunit strongly enhanced the aggregation preventing activity of αβ-NAC pointing to a negative regulatory role of this domain for the NAC chaperone activity in vivo.

Show MeSH
Related in: MedlinePlus