Insights into co-translational protein quality control mechanisms

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KOCH, Miriam, 2012. Insights into co-translational protein quality control mechanisms [Dissertation]. Konstanz: University of Konstanz

@phdthesis{Koch2012Insig-19928, title={Insights into co-translational protein quality control mechanisms}, year={2012}, author={Koch, Miriam}, address={Konstanz}, school={Universität Konstanz} }

Koch, Miriam eng deposit-license 2014-07-24T22:25:04Z 2012 Proteins are synthesized by ribosomes in a vectorial manner from the N- towards the C-terminus. During synthesis only parts of the sequence information are available, therefore stable folding of a folding unit or domain can not occur until the protein is completely synthesized. As the nascent polypeptide chain leaves the ribosomal exit tunnel, it exposes hydrophobic residues and is therefore highly prone for unproductive interactions and aggregation in the cytosol. Therefore, cells evolved systems, which protect proteins from unproductive interactions and provide a platform for folding as well as for removing the nascent polypeptides that are already caught in a non-native or unproductive conformation. In eukaryotes, newly synthesized proteins interact co-translationally with a multitude of different ribosome-bound factors including chaperones such as the conserved heterodimeric polypeptide-associated complex (NAC) and a Hsp40/70 based chaperone system (Ssb /Zuo/ Ssz in yeast). Additionally, the E3 ubiquitin ligase Not4 represents a candidate for a protein involved in co-translational quality control by targeting defective or misfolded nascent chains for degradation by the proteasome. This work contributed the following new insights into this broad and complex topic.<br /><br /><br /><br />1. NAC controls the yield of newly synthesized proteins translated in vitro<br /><br />Recent data from our lab suggest that NAC contributes to the chaperone network, as it was shown that NAC functionally cooperates with Ssb in co-translational protein folding in vivo. However, how NAC may act on newly synthesized proteins is still unknown. Therefore, one goal of this work was to characterize the function of NAC on a molecular level. To investigate the effect of NAC on the synthesis and folding of newly made proteins, an in vitro translation system for the synthesis of model polypeptides with and without NAC was generated. In this work it was shown for the first time that NAC affects the process of translation in vitro. NAC regulates the amount of in vitro translation product and this regulation strongly depends on the ribosome association of NAC. Importantly, NAC has a strong impact on the translation of model proteins and a broad range of yeast proteins. While we could exclude that NAC targets the translation product to the ubiquitin proteasome system, we found a strong influence on the kinetics of translation suggesting that NAC influences initiation as well as elongation of translation by a yet unknown mechanism.<br /><br /><br /><br /><br />2. Analysis of Not4 as a putative ribosome-associated protein quality control system of S. cerevisae<br /><br />The E3 ubiquitin protein ligase Not4 is a component of the conserved eukaryotic Ccr4-Not complex suggested to play an important role in protein and mRNA turnover by its ubiquitin ligase and deadenylation activities, respectively. Not4 has been suggested to target stalled nascent polypeptides for degradation and to ubiquitylate the ribosome-associated complex NAC and thereby regulate its stability and ribosome association. However, we could show that NAC associates with ribosomes independently of Not4. However, this work shows that Not4, Caf1 as well as other members of the Ccr4-Not complex interact with translating ribosomes (polysomes). Ribosome association of Not4 and Caf1 was dependent on the presence of nascent polypeptides, suggesting that Not4, and likely the entire Ccr4-Not complex, senses the presence of nascent peptides and thus may function in co-translational protein quality control. Additionally, experimental data support the idea of a connection of Not4 and Caf1 to the non-stop mRNA decay pathway involving Ski7. Polysome association of Not4 and Caf1 was changed in a ski7Δ strain, and likewise Ski7 binding to the 40S subunit was diminished in not4Δ cells. This led to the assumption that Not4 and the Ccr4-Not complex are part of a co-translational quality control system for defective protein and mRNA. The combination of the E3 ligase activity of Not4 and the deadenylation activity of Ccr4 and Caf1 in one complex, which is localized at the translation machinery, would allow the degradation of the protein via the ubiquitin proteasome system and the mRNA degradation initated by deadenylation in a concerted manner. 2012-08-02T08:09:08Z Koch, Miriam Insights into co-translational protein quality control mechanisms

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