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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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β-NAC and β’-NAC show differences in their C-termini.First, a PSI-BLAST search of the NCBI database was performed. Then the sequences were sorted using CLANS [34] and aligned with the alignment programme muscle [35]. An HMM (http://hmmer.org/) was constructed of the fungi sequences and all sequences were aligned against the HMM. The sequences are shown for a) the N-terminus, b) the NAC-domain and c) the C-terminus of β-NAC and β’-NAC of S. cerevisiae, and of β-NAC from C. elegans and H. sapiens. Amino acids are depicted in the one letter-code. β-NAC of S. cerevisiae could be aligned completely to the β-sequences of all kingdoms, but the end of the C-terminus of β’ from S. cerevisiae could not be aligned with the other sequences and was marked as an insert (small letters at the end of the alignment). Colour legend: orange = small hydrophilics, green = small hydrophobics, red = bases, blue = aromatics and colourless = acids/amides and sulphhydrils.
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pone.0143457.g007: β-NAC and β’-NAC show differences in their C-termini.First, a PSI-BLAST search of the NCBI database was performed. Then the sequences were sorted using CLANS [34] and aligned with the alignment programme muscle [35]. An HMM (http://hmmer.org/) was constructed of the fungi sequences and all sequences were aligned against the HMM. The sequences are shown for a) the N-terminus, b) the NAC-domain and c) the C-terminus of β-NAC and β’-NAC of S. cerevisiae, and of β-NAC from C. elegans and H. sapiens. Amino acids are depicted in the one letter-code. β-NAC of S. cerevisiae could be aligned completely to the β-sequences of all kingdoms, but the end of the C-terminus of β’ from S. cerevisiae could not be aligned with the other sequences and was marked as an insert (small letters at the end of the alignment). Colour legend: orange = small hydrophilics, green = small hydrophobics, red = bases, blue = aromatics and colourless = acids/amides and sulphhydrils.

Mentions: We found major functional differences between αβ-NAC and αβ’-NAC. Only αβ-NAC but not αβ’-NAC (even when expressed at similar levels as αβ-NAC) can suppress all defects found in nacΔssbΔ cells including the high sensitivity against translation inhibitory drugs, ribosomal deficiencies that result in halfmer formation, reduced amounts of 80S particles and polysomes, as well as protein aggregation. This suggests that αβ-NAC is functionally most important for yeast vitality. Our results are in agreement with a recent study by Frydman and colleagues analyzing the nascent interactome of NAC [12]. They showed that αβ-NAC has a preference for ribosomes translating metabolic enzymes as well as secretory and membrane proteins while αβ’-NAC preferentially binds to ribosomes translating mitochondrial or ribosomal proteins. This finding implies different substrate pools of αβ-NAC and αβ’-NAC. Both heterodimers are ribosome-associated by the conserved ribosome-binding motif found in β-NAC as well as in β’-NAC (Figs 1B and 7A). In addition, both subunits possess the conserved NAC domain involved in dimerization (Fig 7B). The two different β-subunits display an overall similarity of 64.3% with an identity of 46.5% on their amino acid level. However, β-NAC and β’-NAC obviously reveal strong differences at their C-terminal ends (Fig 7C) as the similarity of this region is only 30.8% with an identity of 10.3%. Both C-termini are predicted to be rather unstructured, however, the C-terminus of β’-NAC is shorter by 8 amino acid residues compared to β-NAC and the last 16 amino acids show no homology to β-NAC at all (Fig 7C). Moreover, β’-NAC has a lower amount of charged amino acids in its C-terminus: 5 negatively charged residues (Asp + Glu) and 2 positively charged residues (Arg + Lys) compared to β-NAC with 11 negatively charged residues (Asp + Glu) and 4 positively charged residues (Arg + Lys). Thus, we speculate that the diverse C-termini of β- and β’-NAC might be involved in substrate selectivity and thus contribute to the functional differences of αβ-NAC and αβ’-NAC. Interestingly, the C-termini of β-NAC subunits from C. elegans and humans also contain a high number of charged residues and are clearly more similar to yeast β-NAC than to β’-NAC (Fig 7C).


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

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

β-NAC and β’-NAC show differences in their C-termini.First, a PSI-BLAST search of the NCBI database was performed. Then the sequences were sorted using CLANS [34] and aligned with the alignment programme muscle [35]. An HMM (http://hmmer.org/) was constructed of the fungi sequences and all sequences were aligned against the HMM. The sequences are shown for a) the N-terminus, b) the NAC-domain and c) the C-terminus of β-NAC and β’-NAC of S. cerevisiae, and of β-NAC from C. elegans and H. sapiens. Amino acids are depicted in the one letter-code. β-NAC of S. cerevisiae could be aligned completely to the β-sequences of all kingdoms, but the end of the C-terminus of β’ from S. cerevisiae could not be aligned with the other sequences and was marked as an insert (small letters at the end of the alignment). Colour legend: orange = small hydrophilics, green = small hydrophobics, red = bases, blue = aromatics and colourless = acids/amides and sulphhydrils.
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Related In: Results  -  Collection

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

pone.0143457.g007: β-NAC and β’-NAC show differences in their C-termini.First, a PSI-BLAST search of the NCBI database was performed. Then the sequences were sorted using CLANS [34] and aligned with the alignment programme muscle [35]. An HMM (http://hmmer.org/) was constructed of the fungi sequences and all sequences were aligned against the HMM. The sequences are shown for a) the N-terminus, b) the NAC-domain and c) the C-terminus of β-NAC and β’-NAC of S. cerevisiae, and of β-NAC from C. elegans and H. sapiens. Amino acids are depicted in the one letter-code. β-NAC of S. cerevisiae could be aligned completely to the β-sequences of all kingdoms, but the end of the C-terminus of β’ from S. cerevisiae could not be aligned with the other sequences and was marked as an insert (small letters at the end of the alignment). Colour legend: orange = small hydrophilics, green = small hydrophobics, red = bases, blue = aromatics and colourless = acids/amides and sulphhydrils.
Mentions: We found major functional differences between αβ-NAC and αβ’-NAC. Only αβ-NAC but not αβ’-NAC (even when expressed at similar levels as αβ-NAC) can suppress all defects found in nacΔssbΔ cells including the high sensitivity against translation inhibitory drugs, ribosomal deficiencies that result in halfmer formation, reduced amounts of 80S particles and polysomes, as well as protein aggregation. This suggests that αβ-NAC is functionally most important for yeast vitality. Our results are in agreement with a recent study by Frydman and colleagues analyzing the nascent interactome of NAC [12]. They showed that αβ-NAC has a preference for ribosomes translating metabolic enzymes as well as secretory and membrane proteins while αβ’-NAC preferentially binds to ribosomes translating mitochondrial or ribosomal proteins. This finding implies different substrate pools of αβ-NAC and αβ’-NAC. Both heterodimers are ribosome-associated by the conserved ribosome-binding motif found in β-NAC as well as in β’-NAC (Figs 1B and 7A). In addition, both subunits possess the conserved NAC domain involved in dimerization (Fig 7B). The two different β-subunits display an overall similarity of 64.3% with an identity of 46.5% on their amino acid level. However, β-NAC and β’-NAC obviously reveal strong differences at their C-terminal ends (Fig 7C) as the similarity of this region is only 30.8% with an identity of 10.3%. Both C-termini are predicted to be rather unstructured, however, the C-terminus of β’-NAC is shorter by 8 amino acid residues compared to β-NAC and the last 16 amino acids show no homology to β-NAC at all (Fig 7C). Moreover, β’-NAC has a lower amount of charged amino acids in its C-terminus: 5 negatively charged residues (Asp + Glu) and 2 positively charged residues (Arg + Lys) compared to β-NAC with 11 negatively charged residues (Asp + Glu) and 4 positively charged residues (Arg + Lys). Thus, we speculate that the diverse C-termini of β- and β’-NAC might be involved in substrate selectivity and thus contribute to the functional differences of αβ-NAC and αβ’-NAC. Interestingly, the C-termini of β-NAC subunits from C. elegans and humans also contain a high number of charged residues and are clearly more similar to yeast β-NAC than to β’-NAC (Fig 7C).

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