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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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NAC is not coregulated with genes encoding ribosomal proteins.a) X-axis: Relative mRNA levels of indicated genes and time points compared to timepoint zero (t = 0, before glucose addition) and normalized to an internal control (housekeeping gene). Cells were harvested at 0 min, 30 min and 60 min after glucose addition and mRNA was extracted. cDNA was obtained by reverse transcription and used for qRT-PCR. b) Serial dilutions of wild type (wt) and chaperone mutant cells were spotted on YPD plates and plates containing the indicated drugs for growth analysis. When cells were plated on the arginine analogue L-canavanine, arginine was omitted. The cells were incubated for 3 days at 30°C. c) Polysome profiles of wt and mutant cells. 10 A260 units of lysates of indicated yeast strains were loaded onto 15–45% linear sucrose gradients as shown in Fig 3. The profiles are representative for three independent runs.
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pone.0143457.g005: NAC is not coregulated with genes encoding ribosomal proteins.a) X-axis: Relative mRNA levels of indicated genes and time points compared to timepoint zero (t = 0, before glucose addition) and normalized to an internal control (housekeeping gene). Cells were harvested at 0 min, 30 min and 60 min after glucose addition and mRNA was extracted. cDNA was obtained by reverse transcription and used for qRT-PCR. b) Serial dilutions of wild type (wt) and chaperone mutant cells were spotted on YPD plates and plates containing the indicated drugs for growth analysis. When cells were plated on the arginine analogue L-canavanine, arginine was omitted. The cells were incubated for 3 days at 30°C. c) Polysome profiles of wt and mutant cells. 10 A260 units of lysates of indicated yeast strains were loaded onto 15–45% linear sucrose gradients as shown in Fig 3. The profiles are representative for three independent runs.

Mentions: The expression of genes encoding proteins involved in ribosome biogenesis is often coregulated with genes coding for ribosomal proteins [24]. This has been reported also for the ribosome-associated chaperone Ssb [25]. As the ribosomal biogenesis and translation defects of cells lacking NAC and Ssb are more pronounced than in the cells lacking only Ssb, we wondered whether the genes EGD1, BTT1 and EGD2 coding for NAC are also coregulated with ribosomal genes. In a previous study from Albanèse et al. [26] where transcriptional analysis of gene expression in response to environmental stress, e.g. heat shock or nitrogene depletion, was performed, NAC was found to be corepressed together with components of the translational apparatus and ribosome biogenesis chaperones such as Ssb and RAC. To further address this question under non-stress conditions, wt cells were grown in medium containing glycerol as carbon source until they reached an OD600 of 0.6 (time point zero). Then the cells were washed and transferred into medium containing glucose because ribosomal genes are upregulated upon carbon upshift from glycerol- to glucose-containing medium. Total RNA was isolated after various time points and followed by quantitative real-time PCR. We found, in agreement with earlier studies [25], that the mRNAs of SSB1 and the ribosomal protein RPL5 as well as the mRNA of the ribosome biogenesis factor JJJ1 were upregulated about 2- to 3.5-fold upon carbon shift (Fig 5A). However, no significantly enhanced transcription of mRNA coding for any of the three NAC subunits was detected. The mRNA levels of EGD1 and EGD2 remained almost constant in comparison to SSB1 or JJJ1 and the mRNA level of BTT1 was even slightly reduced upon carbon shift (Fig 5A). Hence, NAC is not coregulated with ribosomal proteins under these conditions, which is a typical characteristic for ribosomal biogenesis factors and chaperones directly involved in this process, such as Jjj1 or Ssb. It is known that loss of Jjj1 causes a slow growth phenotype and the combined deletion of the SSB1,2 genes and JJJ1 results in synthetic lethality [18]. To further investigate the role of NAC in ribosome biogenesis, we generated jjj1Δ and nacΔjjj1Δ knockout strains to test for a genetic interaction. The nacΔjjj1Δ cells lacking Jjj1 and all three genes encoding NAC showed no synthetic growth phenotype compared to jjj1Δ cells under the conditions tested (Fig 5B). Ribosome profiles of jjj1Δ and nacΔjjj1Δ cells (Fig 5C–5F) revealed that the deletion of JJJ1 resulted in a decrease of 60S subunits and in the appearance of halfmers (Fig 5E), indicating that this strain has a ribosome biogenesis defect as described previously [27, 28]. Loss of NAC in jjj1Δ cells did neither enhance the halfmer formation nor cause a further reduction of 60S, 80S or polysome peaks. This suggests that NAC and Jjj1 do not display overlapping functions in ribosome biogenesis and indicates together with the lack of transcriptional coregulation that NAC supports the activity of the translation apparatus by a mechanism distinct from classical ribosome biogenesis factors.


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

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

NAC is not coregulated with genes encoding ribosomal proteins.a) X-axis: Relative mRNA levels of indicated genes and time points compared to timepoint zero (t = 0, before glucose addition) and normalized to an internal control (housekeeping gene). Cells were harvested at 0 min, 30 min and 60 min after glucose addition and mRNA was extracted. cDNA was obtained by reverse transcription and used for qRT-PCR. b) Serial dilutions of wild type (wt) and chaperone mutant cells were spotted on YPD plates and plates containing the indicated drugs for growth analysis. When cells were plated on the arginine analogue L-canavanine, arginine was omitted. The cells were incubated for 3 days at 30°C. c) Polysome profiles of wt and mutant cells. 10 A260 units of lysates of indicated yeast strains were loaded onto 15–45% linear sucrose gradients as shown in Fig 3. The profiles are representative for three independent runs.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4664479&req=5

pone.0143457.g005: NAC is not coregulated with genes encoding ribosomal proteins.a) X-axis: Relative mRNA levels of indicated genes and time points compared to timepoint zero (t = 0, before glucose addition) and normalized to an internal control (housekeeping gene). Cells were harvested at 0 min, 30 min and 60 min after glucose addition and mRNA was extracted. cDNA was obtained by reverse transcription and used for qRT-PCR. b) Serial dilutions of wild type (wt) and chaperone mutant cells were spotted on YPD plates and plates containing the indicated drugs for growth analysis. When cells were plated on the arginine analogue L-canavanine, arginine was omitted. The cells were incubated for 3 days at 30°C. c) Polysome profiles of wt and mutant cells. 10 A260 units of lysates of indicated yeast strains were loaded onto 15–45% linear sucrose gradients as shown in Fig 3. The profiles are representative for three independent runs.
Mentions: The expression of genes encoding proteins involved in ribosome biogenesis is often coregulated with genes coding for ribosomal proteins [24]. This has been reported also for the ribosome-associated chaperone Ssb [25]. As the ribosomal biogenesis and translation defects of cells lacking NAC and Ssb are more pronounced than in the cells lacking only Ssb, we wondered whether the genes EGD1, BTT1 and EGD2 coding for NAC are also coregulated with ribosomal genes. In a previous study from Albanèse et al. [26] where transcriptional analysis of gene expression in response to environmental stress, e.g. heat shock or nitrogene depletion, was performed, NAC was found to be corepressed together with components of the translational apparatus and ribosome biogenesis chaperones such as Ssb and RAC. To further address this question under non-stress conditions, wt cells were grown in medium containing glycerol as carbon source until they reached an OD600 of 0.6 (time point zero). Then the cells were washed and transferred into medium containing glucose because ribosomal genes are upregulated upon carbon upshift from glycerol- to glucose-containing medium. Total RNA was isolated after various time points and followed by quantitative real-time PCR. We found, in agreement with earlier studies [25], that the mRNAs of SSB1 and the ribosomal protein RPL5 as well as the mRNA of the ribosome biogenesis factor JJJ1 were upregulated about 2- to 3.5-fold upon carbon shift (Fig 5A). However, no significantly enhanced transcription of mRNA coding for any of the three NAC subunits was detected. The mRNA levels of EGD1 and EGD2 remained almost constant in comparison to SSB1 or JJJ1 and the mRNA level of BTT1 was even slightly reduced upon carbon shift (Fig 5A). Hence, NAC is not coregulated with ribosomal proteins under these conditions, which is a typical characteristic for ribosomal biogenesis factors and chaperones directly involved in this process, such as Jjj1 or Ssb. It is known that loss of Jjj1 causes a slow growth phenotype and the combined deletion of the SSB1,2 genes and JJJ1 results in synthetic lethality [18]. To further investigate the role of NAC in ribosome biogenesis, we generated jjj1Δ and nacΔjjj1Δ knockout strains to test for a genetic interaction. The nacΔjjj1Δ cells lacking Jjj1 and all three genes encoding NAC showed no synthetic growth phenotype compared to jjj1Δ cells under the conditions tested (Fig 5B). Ribosome profiles of jjj1Δ and nacΔjjj1Δ cells (Fig 5C–5F) revealed that the deletion of JJJ1 resulted in a decrease of 60S subunits and in the appearance of halfmers (Fig 5E), indicating that this strain has a ribosome biogenesis defect as described previously [27, 28]. Loss of NAC in jjj1Δ cells did neither enhance the halfmer formation nor cause a further reduction of 60S, 80S or polysome peaks. This suggests that NAC and Jjj1 do not display overlapping functions in ribosome biogenesis and indicates together with the lack of transcriptional coregulation that NAC supports the activity of the translation apparatus by a mechanism distinct from classical ribosome biogenesis factors.

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