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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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Ribosome-associated chaperones from S. cerevisiae.a) The Hsp70/Hsp40-chaperone system that consists of RAC (Ssz and Zuo), shown in purple and light green, and Ssb, shown in light blue, forms a functional triad at the ribosome. In addition, β-NAC (shown in blue) and α-NAC (shown in red) that contains a C-terminal UBA (ubiquitin-associated) domain constitute the stable heterodimeric αβ-NAC complex which binds to the ribosome via the ribosome-binding motif in the β-subunit. Both, NAC and Ssb can interact directly with the nascent chain. b) Schematic representation of the different NAC subunits. α-NAC (shown in red) contains a NAC domain and a UBA domain. Besides the NAC domain the two different β-subunits (shown in light and dark blue) also contain a conserved ribosome-binding motif present in their N-termini. c) Schematic drawing of the two NAC mutants investigated in this study. αΔUBA-NAC (shown in red) lacks the C-terminal UBA domain and part of the linker region. In βRRK/AAA-NAC (shown in blue) the conserved RRK-(X)2-KK motif was mutated to AAA-(X)2-KK to abolish ribosome binding.
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pone.0143457.g001: Ribosome-associated chaperones from S. cerevisiae.a) The Hsp70/Hsp40-chaperone system that consists of RAC (Ssz and Zuo), shown in purple and light green, and Ssb, shown in light blue, forms a functional triad at the ribosome. In addition, β-NAC (shown in blue) and α-NAC (shown in red) that contains a C-terminal UBA (ubiquitin-associated) domain constitute the stable heterodimeric αβ-NAC complex which binds to the ribosome via the ribosome-binding motif in the β-subunit. Both, NAC and Ssb can interact directly with the nascent chain. b) Schematic representation of the different NAC subunits. α-NAC (shown in red) contains a NAC domain and a UBA domain. Besides the NAC domain the two different β-subunits (shown in light and dark blue) also contain a conserved ribosome-binding motif present in their N-termini. c) Schematic drawing of the two NAC mutants investigated in this study. αΔUBA-NAC (shown in red) lacks the C-terminal UBA domain and part of the linker region. In βRRK/AAA-NAC (shown in blue) the conserved RRK-(X)2-KK motif was mutated to AAA-(X)2-KK to abolish ribosome binding.

Mentions: The folding of newly synthesized proteins requires the assistance of molecular chaperones. At the forefront are ribosome-associated chaperones, which contact nascent polypeptides to control early protein folding processes and to prevent aggregation or degradation of newly synthesized proteins [1, 2]. Yeast ribosomes are transiently associated with two different types of chaperone systems. One is a Hsp70/Hsp40-based chaperone system consisting of the ribosome-associated complex (RAC), a heterodimer formed by Zuo(tin) and Ssz, and Ssb. The second system is the nascent polypeptide-associated complex (NAC). Both systems bind transiently to the large ribosomal subunit for interaction with nascent polypeptides early during protein biogenesis (Fig 1A).


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

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

Ribosome-associated chaperones from S. cerevisiae.a) The Hsp70/Hsp40-chaperone system that consists of RAC (Ssz and Zuo), shown in purple and light green, and Ssb, shown in light blue, forms a functional triad at the ribosome. In addition, β-NAC (shown in blue) and α-NAC (shown in red) that contains a C-terminal UBA (ubiquitin-associated) domain constitute the stable heterodimeric αβ-NAC complex which binds to the ribosome via the ribosome-binding motif in the β-subunit. Both, NAC and Ssb can interact directly with the nascent chain. b) Schematic representation of the different NAC subunits. α-NAC (shown in red) contains a NAC domain and a UBA domain. Besides the NAC domain the two different β-subunits (shown in light and dark blue) also contain a conserved ribosome-binding motif present in their N-termini. c) Schematic drawing of the two NAC mutants investigated in this study. αΔUBA-NAC (shown in red) lacks the C-terminal UBA domain and part of the linker region. In βRRK/AAA-NAC (shown in blue) the conserved RRK-(X)2-KK motif was mutated to AAA-(X)2-KK to abolish ribosome binding.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0143457.g001: Ribosome-associated chaperones from S. cerevisiae.a) The Hsp70/Hsp40-chaperone system that consists of RAC (Ssz and Zuo), shown in purple and light green, and Ssb, shown in light blue, forms a functional triad at the ribosome. In addition, β-NAC (shown in blue) and α-NAC (shown in red) that contains a C-terminal UBA (ubiquitin-associated) domain constitute the stable heterodimeric αβ-NAC complex which binds to the ribosome via the ribosome-binding motif in the β-subunit. Both, NAC and Ssb can interact directly with the nascent chain. b) Schematic representation of the different NAC subunits. α-NAC (shown in red) contains a NAC domain and a UBA domain. Besides the NAC domain the two different β-subunits (shown in light and dark blue) also contain a conserved ribosome-binding motif present in their N-termini. c) Schematic drawing of the two NAC mutants investigated in this study. αΔUBA-NAC (shown in red) lacks the C-terminal UBA domain and part of the linker region. In βRRK/AAA-NAC (shown in blue) the conserved RRK-(X)2-KK motif was mutated to AAA-(X)2-KK to abolish ribosome binding.
Mentions: The folding of newly synthesized proteins requires the assistance of molecular chaperones. At the forefront are ribosome-associated chaperones, which contact nascent polypeptides to control early protein folding processes and to prevent aggregation or degradation of newly synthesized proteins [1, 2]. Yeast ribosomes are transiently associated with two different types of chaperone systems. One is a Hsp70/Hsp40-based chaperone system consisting of the ribosome-associated complex (RAC), a heterodimer formed by Zuo(tin) and Ssz, and Ssb. The second system is the nascent polypeptide-associated complex (NAC). Both systems bind transiently to the large ribosomal subunit for interaction with nascent polypeptides early during protein biogenesis (Fig 1A).

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