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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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Halfmer formation of nacΔssbΔ knockout cells can be prevented by expression of αβ-NAC.a-i) Polysome profiles derived from wild type (wt) or mutant yeast cells. Absorbance traces at 254 nm are shown. Cells were grown to an optical density (OD600) of 0.8 in SD-Ura medium. 10 A260 units of lysates of indicated cells were loaded onto 15–45% linear sucrose gradients to isolate ribosomal fractions (40S, 60S, 80S and polysomes) as indicated by centrifugation and subsequent fractionation. Polysome profiles show: a) wt + empty vector (ev), b) ssbΔ cells + ev, c-i) nacΔssbΔ cells + ev (c), + β-NAC (d), β’-NAC (e), αΔUBAβ-NAC (f), αβ-NAC (g) + αβ’-NAC (h) and αβRRK/AAA-NAC (i) The profiles are representative for three independent experiments.
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pone.0143457.g003: Halfmer formation of nacΔssbΔ knockout cells can be prevented by expression of αβ-NAC.a-i) Polysome profiles derived from wild type (wt) or mutant yeast cells. Absorbance traces at 254 nm are shown. Cells were grown to an optical density (OD600) of 0.8 in SD-Ura medium. 10 A260 units of lysates of indicated cells were loaded onto 15–45% linear sucrose gradients to isolate ribosomal fractions (40S, 60S, 80S and polysomes) as indicated by centrifugation and subsequent fractionation. Polysome profiles show: a) wt + empty vector (ev), b) ssbΔ cells + ev, c-i) nacΔssbΔ cells + ev (c), + β-NAC (d), β’-NAC (e), αΔUBAβ-NAC (f), αβ-NAC (g) + αβ’-NAC (h) and αβRRK/AAA-NAC (i) The profiles are representative for three independent experiments.

Mentions: Previous studies revealed that nacΔssbΔ cells show a defect in ribosome biogenesis leading to the formation of ribosomal halfmers and a reduced translational activity [18]. This defect in ribosome biogenesis can be investigated by separating total cell lysate on a sucrose gradient using ultracentrifugation and subsequent fractionation of the gradient monitoring ribosomal species by measuring the absorption at 254 nm. The peak heights of the absorption traces detected at 254 nm could be used as sensitive indicators for the levels of each ribosomal species because equal absorption units of the samples were loaded. Thereby, the shoulder in the 80S and polysome peaks of the double knockout cells represents the presence of ribosomal halfmers in such fractionation experiments (Fig 3C, arrows). Such halfmers consist of an uncomplexed 40S subunit bound to the mRNA and are typically caused by an impaired balance of 40S and 60S ribosomal subunits due to defects in the assembly of 60S particles [22, 23]. Indeed, higher levels of 40S subunits were detected in nacΔssbΔ cells compared to the wild type. Moreover, the 80S monosome and polysome peaks were significantly reduced and ribosomal halfmers were present in nacΔssbΔ cells compared to wt cells indicating the reduced translational activity (Fig 3A and 3C). As reported earlier [18], these ribosomal defects are clearly less pronounced in cells lacking only Ssb (Fig 3B).


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

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

Halfmer formation of nacΔssbΔ knockout cells can be prevented by expression of αβ-NAC.a-i) Polysome profiles derived from wild type (wt) or mutant yeast cells. Absorbance traces at 254 nm are shown. Cells were grown to an optical density (OD600) of 0.8 in SD-Ura medium. 10 A260 units of lysates of indicated cells were loaded onto 15–45% linear sucrose gradients to isolate ribosomal fractions (40S, 60S, 80S and polysomes) as indicated by centrifugation and subsequent fractionation. Polysome profiles show: a) wt + empty vector (ev), b) ssbΔ cells + ev, c-i) nacΔssbΔ cells + ev (c), + β-NAC (d), β’-NAC (e), αΔUBAβ-NAC (f), αβ-NAC (g) + αβ’-NAC (h) and αβRRK/AAA-NAC (i) The profiles are representative for three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0143457.g003: Halfmer formation of nacΔssbΔ knockout cells can be prevented by expression of αβ-NAC.a-i) Polysome profiles derived from wild type (wt) or mutant yeast cells. Absorbance traces at 254 nm are shown. Cells were grown to an optical density (OD600) of 0.8 in SD-Ura medium. 10 A260 units of lysates of indicated cells were loaded onto 15–45% linear sucrose gradients to isolate ribosomal fractions (40S, 60S, 80S and polysomes) as indicated by centrifugation and subsequent fractionation. Polysome profiles show: a) wt + empty vector (ev), b) ssbΔ cells + ev, c-i) nacΔssbΔ cells + ev (c), + β-NAC (d), β’-NAC (e), αΔUBAβ-NAC (f), αβ-NAC (g) + αβ’-NAC (h) and αβRRK/AAA-NAC (i) The profiles are representative for three independent experiments.
Mentions: Previous studies revealed that nacΔssbΔ cells show a defect in ribosome biogenesis leading to the formation of ribosomal halfmers and a reduced translational activity [18]. This defect in ribosome biogenesis can be investigated by separating total cell lysate on a sucrose gradient using ultracentrifugation and subsequent fractionation of the gradient monitoring ribosomal species by measuring the absorption at 254 nm. The peak heights of the absorption traces detected at 254 nm could be used as sensitive indicators for the levels of each ribosomal species because equal absorption units of the samples were loaded. Thereby, the shoulder in the 80S and polysome peaks of the double knockout cells represents the presence of ribosomal halfmers in such fractionation experiments (Fig 3C, arrows). Such halfmers consist of an uncomplexed 40S subunit bound to the mRNA and are typically caused by an impaired balance of 40S and 60S ribosomal subunits due to defects in the assembly of 60S particles [22, 23]. Indeed, higher levels of 40S subunits were detected in nacΔssbΔ cells compared to the wild type. Moreover, the 80S monosome and polysome peaks were significantly reduced and ribosomal halfmers were present in nacΔssbΔ cells compared to wt cells indicating the reduced translational activity (Fig 3A and 3C). As reported earlier [18], these ribosomal defects are clearly less pronounced in cells lacking only Ssb (Fig 3B).

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