Insights into functions and mechanisms of ribosome-associated chaperones from Saccharomyces cerevisiae

II. INTRODUCTION 6 II.1. The ribosome 6 II.1.1 The homeostasis of ribosomes 7 II.1.2 The ribosome structure 8 II.1.3 Ribosomal tunnel exit and ribosome-associated factors 10 II.2 Protein folding 11 II.2.1 Protein folding in the cell 12 II.2.2 De novo protein folding: co-vs. post-translational protein folding 13 II.2.3 Molecular chaperones 15-The Hsp70 family 16-J-protein Hsp40s 17-Nucleotide exchange factors 17 II.3 Ribosome-associated chaperones 19 II.3.1 Ribosome-associated chaperones and de novo folding in the E. coli cytosol 20 II.3.2 Ribosome-associated chaperones and de novo folding in the yeast cytosol 22-The nascent chain-associated complex (NAC) 23-The Ssb/Ssz/Zuo chaperone triad 24 The folding of newly synthesized proteins into their native structures is a fundamental but failure prone process and therefore controlled by a network of molecular chaperones to assist and ensure correct protein folding events. Chaperones that guide the folding of newly synthesized proteins in the cytosol are classified into two groups: chaperones that are recruited to the ribosome are the first interaction partners of newborn proteins. They are assumed to protect nascent polypeptides against harmful conditions and to support initial co-translational folding steps, while cytosolic chaperones act subsequently on a subset of newly synthesized proteins to mainly promote post-translational folding steps. Chaperones associating with the ribosome have been found in all kingdoms of life. While the Trigger Factor (TF) is the only known ribosome-associated chaperone in bacteria, two systems exist in yeast and higher eukaryotes. Both systems, the nascent chain-associated complex (NAC) and the yeast tripartite Ssb-system (Ssb/RAC) are unrelated to TF. In contrast to bacterial TF, the functions of ribosome-associated systems in yeast are still barely defined. The Hsp70/40-based Ssb/RAC-system is per definition a canonical chaperone system, however, its precise function and potential substrates are unknown. Moreover, it is unclear whether NAC contributes to the folding network for newly synthesized proteins since that far no function of NAC could be unambiguously assigned. This work adds to the knowledge about ribosome-associated chaperones in yeast and focuses on the functions and mechanisms of NAC and the Ssb/RAC-system during de novo protein folding. Genetic surveys were combined with biochemical approaches in order to gain insights into the complex cytosolic chaperone network of eukaryotes. The key-results are summarized below: • The first main contribution of this study is the finding that the Ssb-system functionally cooperates with NAC in co-translational protein folding in vivo. Simultaneous deletions of genes encoding NAC and Ssb caused a severe synthetic sickness …