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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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Expression levels of β-NAC are important for complementation of nacΔssbΔ halfmers.Polysome profiling with wild type (wt) or mutant yeast cells. Absorbance traces at 254 nm are shown. 10 A260 units of lysates of indicated yeast cells were loaded onto 15–45% linear sucrose gradients similar to Fig 3. a) Polysome profile of nacΔssbΔ cells + empty vector control (ev) (in grey). b) and c) Complementation of nacΔssbΔ cells with promoter-swapped β-NAC constructs alone. d) nacΔssbΔ cells expressing α-NAC alone. e) and f) Polysome profiles of nacΔssbΔ cells expressing promoter-swapped β-NAC constructs in combination with α-NAC. Arrows indicate halfmers. The profiles are representative for three independent runs.
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pone.0143457.g004: Expression levels of β-NAC are important for complementation of nacΔssbΔ halfmers.Polysome profiling with wild type (wt) or mutant yeast cells. Absorbance traces at 254 nm are shown. 10 A260 units of lysates of indicated yeast cells were loaded onto 15–45% linear sucrose gradients similar to Fig 3. a) Polysome profile of nacΔssbΔ cells + empty vector control (ev) (in grey). b) and c) Complementation of nacΔssbΔ cells with promoter-swapped β-NAC constructs alone. d) nacΔssbΔ cells expressing α-NAC alone. e) and f) Polysome profiles of nacΔssbΔ cells expressing promoter-swapped β-NAC constructs in combination with α-NAC. Arrows indicate halfmers. The profiles are representative for three independent runs.

Mentions: To test which subunit(s) of NAC complement(s) the ribosomal defects, ribosome profiles were generated from nacΔssbΔ cells expressing different NAC variants. We found that in contrast to the growth analysis, only the expression of the αβ-NAC heterodimer could restore the ribosome biogenesis defects observed in nacΔssbΔ cells (Fig 3G). This is demonstrated by a decreased amount of halfmers, a reduced 40S peak and enhanced 80S and polysome peaks resulting in a profile that is similar to ssbΔ cells (Fig 3B, 3C and 3G). Importantly, expression of the ribosome-binding deficient αβRRK/AAA-NAC version did not suppress these deficiencies in ribosome biogenesis and translation (Fig 3I). Moreover, neither expression of β-NAC nor β’-NAC alone cured the ribosomal defects (Fig 3D and 3E). A small reduction in the amount of halfmers could be observed upon expression of αβ’-NAC (Fig 3H). We also investigated whether the expression levels of β-NAC and β’-NAC are crucial for the suppression of ribosomal defects in nacΔssbΔ cells (Fig 4). High level expression of β’-NAC driven by the EGD1 promoter and terminator elements with or without coexpression of α-NAC resulted also in a very mild reduction of ribosomal halfmers confirming again that the β’-NAC subunit and consequently also the αβ’-NAC heterodimer are functionally distinct from β-NAC and αβ-NAC, respectively, even when expressed at similar levels (Fig 4C and 4F). Moreover, a reduced expression of αβ-NAC did not complement the aberrant translation phenotype (Fig 4B and 4E) and also α-NAC itself could not prevent halfmer formation of nacΔssbΔ knockout cells (Fig 4D). Interestingly, the expression of the αΔUBAβ-NAC mutant version in nacΔssbΔ cells (Fig 3F) reduced the halfmer formation whereas the 80S and polysome peaks were still reduced compared to ssbΔ cells, suggesting that the UBA domain functionally contributes to the function of αβ-NAC in translation.


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

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

Expression levels of β-NAC are important for complementation of nacΔssbΔ halfmers.Polysome profiling with wild type (wt) or mutant yeast cells. Absorbance traces at 254 nm are shown. 10 A260 units of lysates of indicated yeast cells were loaded onto 15–45% linear sucrose gradients similar to Fig 3. a) Polysome profile of nacΔssbΔ cells + empty vector control (ev) (in grey). b) and c) Complementation of nacΔssbΔ cells with promoter-swapped β-NAC constructs alone. d) nacΔssbΔ cells expressing α-NAC alone. e) and f) Polysome profiles of nacΔssbΔ cells expressing promoter-swapped β-NAC constructs in combination with α-NAC. Arrows indicate halfmers. The profiles are representative for three independent runs.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664479&req=5

pone.0143457.g004: Expression levels of β-NAC are important for complementation of nacΔssbΔ halfmers.Polysome profiling with wild type (wt) or mutant yeast cells. Absorbance traces at 254 nm are shown. 10 A260 units of lysates of indicated yeast cells were loaded onto 15–45% linear sucrose gradients similar to Fig 3. a) Polysome profile of nacΔssbΔ cells + empty vector control (ev) (in grey). b) and c) Complementation of nacΔssbΔ cells with promoter-swapped β-NAC constructs alone. d) nacΔssbΔ cells expressing α-NAC alone. e) and f) Polysome profiles of nacΔssbΔ cells expressing promoter-swapped β-NAC constructs in combination with α-NAC. Arrows indicate halfmers. The profiles are representative for three independent runs.
Mentions: To test which subunit(s) of NAC complement(s) the ribosomal defects, ribosome profiles were generated from nacΔssbΔ cells expressing different NAC variants. We found that in contrast to the growth analysis, only the expression of the αβ-NAC heterodimer could restore the ribosome biogenesis defects observed in nacΔssbΔ cells (Fig 3G). This is demonstrated by a decreased amount of halfmers, a reduced 40S peak and enhanced 80S and polysome peaks resulting in a profile that is similar to ssbΔ cells (Fig 3B, 3C and 3G). Importantly, expression of the ribosome-binding deficient αβRRK/AAA-NAC version did not suppress these deficiencies in ribosome biogenesis and translation (Fig 3I). Moreover, neither expression of β-NAC nor β’-NAC alone cured the ribosomal defects (Fig 3D and 3E). A small reduction in the amount of halfmers could be observed upon expression of αβ’-NAC (Fig 3H). We also investigated whether the expression levels of β-NAC and β’-NAC are crucial for the suppression of ribosomal defects in nacΔssbΔ cells (Fig 4). High level expression of β’-NAC driven by the EGD1 promoter and terminator elements with or without coexpression of α-NAC resulted also in a very mild reduction of ribosomal halfmers confirming again that the β’-NAC subunit and consequently also the αβ’-NAC heterodimer are functionally distinct from β-NAC and αβ-NAC, respectively, even when expressed at similar levels (Fig 4C and 4F). Moreover, a reduced expression of αβ-NAC did not complement the aberrant translation phenotype (Fig 4B and 4E) and also α-NAC itself could not prevent halfmer formation of nacΔssbΔ knockout cells (Fig 4D). Interestingly, the expression of the αΔUBAβ-NAC mutant version in nacΔssbΔ cells (Fig 3F) reduced the halfmer formation whereas the 80S and polysome peaks were still reduced compared to ssbΔ cells, suggesting that the UBA domain functionally contributes to the function of αβ-NAC in translation.

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