Limits...
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.

Show MeSH

Related in: MedlinePlus

The αβ-NAC complex and β-NAC under control of their endogenous promoter complement the growth defect of nacΔssbΔ cells.a) Schematic drawing of the different plasmid-encoded NAC constructs used in this study. Plasmids encoding wild type (wt) and mutant αβ-NAC, either alone or in complex, were cloned in the vector backbone pRS316 reported by [18]. b) Growth analysis of wt and mutant yeast cells expressing different NAC versions from plasmids as indicated. Serial dilutions were spotted on synthetic complete media without uracil (SD-Ura) containing the indicated drugs. When cells were plated on the arginine analogue L-canavanine, arginine was omitted. The cells were incubated for 3 days at 30°C. c) The promoter (P)—and terminator (T)- regions of EGD1 were replaced with the corresponding regions of BTT1 and vice versa and cloned in the vector backbone of pRS316 with or without EGD2. BTT1 under its endogenous promoter and terminator was also cloned into pRS316 together with EGD2. d) Growth analyses were performed as described in b).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4664479&req=5

pone.0143457.g002: The αβ-NAC complex and β-NAC under control of their endogenous promoter complement the growth defect of nacΔssbΔ cells.a) Schematic drawing of the different plasmid-encoded NAC constructs used in this study. Plasmids encoding wild type (wt) and mutant αβ-NAC, either alone or in complex, were cloned in the vector backbone pRS316 reported by [18]. b) Growth analysis of wt and mutant yeast cells expressing different NAC versions from plasmids as indicated. Serial dilutions were spotted on synthetic complete media without uracil (SD-Ura) containing the indicated drugs. When cells were plated on the arginine analogue L-canavanine, arginine was omitted. The cells were incubated for 3 days at 30°C. c) The promoter (P)—and terminator (T)- regions of EGD1 were replaced with the corresponding regions of BTT1 and vice versa and cloned in the vector backbone of pRS316 with or without EGD2. BTT1 under its endogenous promoter and terminator was also cloned into pRS316 together with EGD2. d) Growth analyses were performed as described in b).

Mentions: The loss of NAC does not result in a growth phenotype, while nacΔssbΔ cells lacking all NAC and Ssb chaperone encoding genes (EGD1Δ, EGD2Δ, BTT1Δ, SSB1Δ, SSB2Δ) show a strong growth deficiency compared to wild type (wt) or ssbΔ cells at 30°C, in particular in the presence of drugs which impair protein synthesis or folding like the arginine analogue L-canavanine or the translation inhibitor hygromycin B [18]. To understand which NAC subunits are essential for growth, we expressed NAC subunits encoded on centromeric plasmids (Fig 2A) individually or in combinations and tested their ability to complement the growth defects of nacΔssbΔ cells (Fig 2B). Expression of NAC genes was driven by the respective authentic promoter (Fig 2A) and protein levels were probed by Western blotting revealing similar expression levels as in the wt (S2B Fig).


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

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

The αβ-NAC complex and β-NAC under control of their endogenous promoter complement the growth defect of nacΔssbΔ cells.a) Schematic drawing of the different plasmid-encoded NAC constructs used in this study. Plasmids encoding wild type (wt) and mutant αβ-NAC, either alone or in complex, were cloned in the vector backbone pRS316 reported by [18]. b) Growth analysis of wt and mutant yeast cells expressing different NAC versions from plasmids as indicated. Serial dilutions were spotted on synthetic complete media without uracil (SD-Ura) containing the indicated drugs. When cells were plated on the arginine analogue L-canavanine, arginine was omitted. The cells were incubated for 3 days at 30°C. c) The promoter (P)—and terminator (T)- regions of EGD1 were replaced with the corresponding regions of BTT1 and vice versa and cloned in the vector backbone of pRS316 with or without EGD2. BTT1 under its endogenous promoter and terminator was also cloned into pRS316 together with EGD2. d) Growth analyses were performed as described in b).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0143457.g002: The αβ-NAC complex and β-NAC under control of their endogenous promoter complement the growth defect of nacΔssbΔ cells.a) Schematic drawing of the different plasmid-encoded NAC constructs used in this study. Plasmids encoding wild type (wt) and mutant αβ-NAC, either alone or in complex, were cloned in the vector backbone pRS316 reported by [18]. b) Growth analysis of wt and mutant yeast cells expressing different NAC versions from plasmids as indicated. Serial dilutions were spotted on synthetic complete media without uracil (SD-Ura) containing the indicated drugs. When cells were plated on the arginine analogue L-canavanine, arginine was omitted. The cells were incubated for 3 days at 30°C. c) The promoter (P)—and terminator (T)- regions of EGD1 were replaced with the corresponding regions of BTT1 and vice versa and cloned in the vector backbone of pRS316 with or without EGD2. BTT1 under its endogenous promoter and terminator was also cloned into pRS316 together with EGD2. d) Growth analyses were performed as described in b).
Mentions: The loss of NAC does not result in a growth phenotype, while nacΔssbΔ cells lacking all NAC and Ssb chaperone encoding genes (EGD1Δ, EGD2Δ, BTT1Δ, SSB1Δ, SSB2Δ) show a strong growth deficiency compared to wild type (wt) or ssbΔ cells at 30°C, in particular in the presence of drugs which impair protein synthesis or folding like the arginine analogue L-canavanine or the translation inhibitor hygromycin B [18]. To understand which NAC subunits are essential for growth, we expressed NAC subunits encoded on centromeric plasmids (Fig 2A) individually or in combinations and tested their ability to complement the growth defects of nacΔssbΔ cells (Fig 2B). Expression of NAC genes was driven by the respective authentic promoter (Fig 2A) and protein levels were probed by Western blotting revealing similar expression levels as in the wt (S2B Fig).

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