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

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


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KOPLIN, Ansgar, 2009. Insights into functions and mechanisms of ribosome-associated chaperones from Saccharomyces cerevisiae [Dissertation]. Konstanz: University of Konstanz

@phdthesis{Koplin2009Insig-7327, title={Insights into functions and mechanisms of ribosome-associated chaperones from Saccharomyces cerevisiae}, year={2009}, author={Koplin, Ansgar}, address={Konstanz}, school={Universität Konstanz} }

Einblicke in Funktionen und Mechanismen Ribosom-assoziierter Chaperone in Saccharomyces cerevisiae Insights into functions and mechanisms of ribosome-associated chaperones from Saccharomyces cerevisiae 2011-07-31T22:25:03Z 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 cotranslational folding steps, while cytosolic chaperones act subsequently on a subset of newly synthesized proteins to mainly promote post-translational folding steps.<br />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.<br /><br />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.<br /><br />The key-results are summarized below:<br />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 of cells grown at 30¡C and the loss of cell viability under protein folding stress conditions. Deprivation of Ssb impaired NAC association with translating ribosomes and provoked aggregation of newly synthesized proteins, which was enhanced by additional deletion of NAC. Further analysis discovered a second function of Ssb/RAC and NAC in regulating the amount of 60S and 40S ribosomal particles suggesting a profound role in the biogenesis of ribosomes. Nac!ssb! cells revealed the formation of ribosomal halfmers and a pronounced deficiency of ribosomal subunits accompanied by strongly reduced amounts of translating ribosomes. These data provide for the first time evidence that the two ribosome-associated systems NAC and Ssb/RAC are functionally interconnected and contribute to two major cellular processes: the folding of newly synthesized proteins and the production of actively translating ribosomes.<br /><br />The second main contribution of this study is the identification of the genetic and functional interaction of NAC and the Hsp110 Sse1. Sse1 functions as nucleotide exchange factor (NEF) for two types of Hsp70s, the cytosolic Ssa and the ribosome-associated Ssb. This study showed that NAC and Sse1 genetically interact and the simultaneous deletion caused a severe growth impairment at 30¡C, a sensitivity against drugs inducing protein folding stress and a mild induction of the cellular heat shock response. The phenotype could be complemented by expressing NAC or by overexpression of Fes1, the second NEF for Ssa and Ssb-type of Hsp70s. Loss of Sse1 was accompanied by protein aggregation including polyubiquitinated proteins which was enhanced by additional NAC deletion. Mass spectrometry identified 13 potential Sse1 and NAC substrates including the enzyme Glucose-6-phophate-dehydrogenase. These data further support the theory that NAC is a bonafide member of the cytosolic chaperone network.<br /><br />In summary, the findings of this work have led to a clearer picture of the mechanisms and functional interplay of ribosome-associated chaperones as decisive regulators at the birthplace of new proteins. terms-of-use Koplin, Ansgar application/pdf Koplin, Ansgar eng 2009 2011-03-24T17:33:33Z

Dateiabrufe seit 01.10.2014 (Informationen über die Zugriffsstatistik)

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