title: Functional Dissection of the Nascent Polypeptide-Associated Complex in Saccharomyces cerevisiae.
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.
authors.affiliation: Molecular Microbiology, Department of Biology, University of Konstanz, 78457, Konstanz, Germany.|Konstanz Research School of Chemical Biology, University of Konstanz, 78457, Konstanz, Germany.
authors.forename: Ann-Kathrin
authors.initials: AK
authors.lastname: Ott
authors.affiliation: Molecular Microbiology, Department of Biology, University of Konstanz, 78457, Konstanz, Germany.|Konstanz Research School of Chemical Biology, University of Konstanz, 78457, Konstanz, Germany.
authors.forename: Lisa
authors.initials: L
authors.lastname: Locher
authors.affiliation: Molecular Microbiology, Department of Biology, University of Konstanz, 78457, Konstanz, Germany.|Konstanz Research School of Chemical Biology, University of Konstanz, 78457, Konstanz, Germany.
authors.forename: Miriam
authors.initials: M
authors.lastname: Koch
authors.affiliation: Molecular Microbiology, Department of Biology, University of Konstanz, 78457, Konstanz, Germany.
authors.forename: Elke
authors.initials: E
authors.lastname: Deuerling
mesh_terms: D000595:Amino Acid Sequence; D006360:Heat-Shock Proteins / Q000378:metabolism; D018832:Molecular Chaperones / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; C096099:nascent-polypeptide-associated complex