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

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 (nac Delta ssb Delta) 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 (alpha-NAC, beta-NAC and beta'-NAC) and of different NAC heterodimers found in yeast (alpha beta-NAC and alpha beta'-NAC) by analyzing their capability to complement the pleiotropic phenotype of nac Delta ssb Delta cells. We show that the abundant heterodimer alpha beta-NAC but not its paralogue alpha beta'-NAC is able to suppress all phenotypic defects of nac Delta ssb Delta cells including global protein aggregation as well as translation and growth deficiencies. This suggests that alpha beta-NAC and alpha beta'-NAC are functionally distinct from each other. The function of alpha beta-NAC strictly depends on its ribosome association and on its high level of expression. Expression of individual beta-NAC, beta'-NAC or alpha-NAC subunits as well as alpha beta'-NAC ameliorated protein aggregation in nac Delta ssb Delta cells to different extents while only beta-NAC was able to restore growth defects suggesting chaperoning activities for beta-NAC sufficient to decrease the sensitivity of nac Delta ssb Delta cells against L-canavanine or hygromycin B. Interestingly, deletion of the ubiquitin-associated (UBA)-domain of the alpha-NAC subunit strongly enhanced the aggregation preventing activity of alpha beta-NAC pointing to a negative regulatory role of this domain for the NAC chaperone activity in vivo.