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Insights into cotranslational protein folding and protein quality control systems on ribosomes

Insights into cotranslational protein folding and protein quality control systems on ribosomes

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PREISSLER, Steffen, 2011. Insights into cotranslational protein folding and protein quality control systems on ribosomes [Dissertation]. Konstanz: University of Konstanz

@phdthesis{Preiler2011Insig-17414, title={Insights into cotranslational protein folding and protein quality control systems on ribosomes}, year={2011}, author={Preißler, Steffen}, note={Teilweise veröffentlicht in:
Eichmann C.*, Preissler S.*, Riek R., Deuerling E. (2010) PNAS
Merz F., Boehringer D., Schaffitzel C., Preissler S., Hoffmann A., Maier T., Rutkowska A., Lozza J., Ban N., Bukau B., Deuerling E. (2008) EMBO J.
Huber D., Rajagopalan N., Preissler S., Rocco M. A., Merz F., Kramer G., Bukau B. (2011) Molecular Cell
Koplin A., Preissler S., Ilina Y., Koch M., Scior A., Erhardt M., Deuerling E. (2010) JCB
Fiaux J., Horst J., Scior A., Preissler S., Koplin A., Bukau B., Deuerling E. (2010) JBC
Scior A.*, Preissler S.*, Koch M., Deuerling E. (2011) BMC Biotechnology
}, address={Konstanz}, school={Universität Konstanz} }

eng 2011 deposit-license Upon their synthesis by ribosomes, proteins have to fold into unique three-dimensional structures to become biologically active. Understanding the mechanisms by which newly synthesized proteins acquire and maintain their shapes under cellular conditions is therefore particularly important. In addition, many nascent polypeptides become modified, targeted to membranes, or marked for degradation when their synthesis was defective. For this, all cells<br />contain specialized factors that directly act on nascent polypeptide chains. Although not much is known about their functional interplay, these factors are thought to establish a cotranslational quality control network for newly made proteins. This work focused on<br />investigating the principles of de novo protein folding and the cellular strategies to support the fidelity of this process in both, pro- and eukaryotic model organisms. The following results were obtained:<br /><br />I. Analysis of cotranslational protein folding and ribosome-associated factors in Escherichia coli<br />(A) This work revealed for the first time the cotranslational folding pathway of a model<br />nascent polypeptide at the atomic level. In a collaborative effort, NMR spectroscopy was<br />used to monitor the conformation of the SH3 domain from α-spectrin at sequential stages during its synthesis. The translation of SH3 was therefore site-specifically arrested on ribosomes in Escherichia coli cells to generate 15N,13C-labeled nascent polypeptides. To<br />provide snapshots of the translation process, nascent chains were designed to either expose the entire SH3 domain or C-terminally truncated versions thereof. The data showed that nascent SH3 remains unstructured during elongation but adopts a native-like conformation<br />as soon as the entire sequence information is available outside the ribosome. In addition, the ribosome neither imposes conformational constraints nor forms significant contacts with the unfolded nascent SH3 domain. Thus, SH3 folds on ribosomes in a domainwise manner without populating folding intermediates.<br /><br /><br />(B) The molecular interaction between the bacterial ribosome-associated chaperone Trigger<br />Factor (TF) and nascent polypeptides was investigated using a site-specific crosslinking<br />approach. For this, radioactively labeled nascent chains were produced in vitro and<br />subjected to binding experiments with TF. The contribution of this work was to clone DNA<br />templates encoding model nascent chains and to generate a suitable in vitro<br />transcription/translation system, which was derived from Escherichia coli cells, to study the<br />chaperone activity of TF on ribosomes. The analysis revealed that ribosome-bound TF can<br />accommodate nascent chains of different lengths and folding states in its interior to protect<br />them against aggregation or premature degradation. This may explain how TF is able to<br />assist cotranslational folding of a broad spectrum of nascent proteins.<br /><br /><br />(C) It has been suggested that SecA-mediated translocation of secretory proteins across the<br />cytoplasmic membrane of Escherichia coli cells occurs posttranslationally. This work<br />contributed the key finding that SecA associates directly with ribosomes in vitro. Together<br />with other results, a new model was proposed, according to which secretory proteins are<br />recognized already cotranslationally by ribosome-bound SecA to direct them efficiently to the<br />posttranslational translocation pathway.<br /><br /><br />II. Analysis of the ribosome-associated protein quality control system of<br />Saccharomyces cerevisiae<br />(A) The E3 ubiquitin-protein ligase Not4 is a component of the conserved eukaryotic Ccr4-<br />Not complex and has been suggested to target stalled nascent polypeptides for degradation.<br />To investigate whether Not4 plays a role in the ribosome-bound protein quality control<br />system of Saccharomyces cerevisiae, the interaction of Not4 with ribosomes was analyzed.<br />The results showed that Not4 and Caf1, another subunit of the complex, associate with<br />polyribosomes in vivo. Ribosome-association of both factors, however, was dependent on<br />the presence of nascent polypeptides, suggesting that Not4, or the entire Ccr4-Not complex,<br />senses the presence of nascent peptides and thus may function in cotranslational protein<br />quality control. Moreover, deletion of the NOT4 gene caused temperature-dependent<br />aggregation of a broad range of different protein species and the constitutive upregulation of<br />heat-shock responsive reporters, indicating folding stress. These data functionally connect<br />the Ccr4-Not complex to the cellular protein homeostasis network.<br /><br /><br />(B) In yeast, the stable heterodimeric ribosome-associated complex (RAC) is composed of<br />the Hsp70 and Hsp40 chaperones Ssz and Zuotin, respectively. RAC acts as cochaperone<br />for the Hsp70 chaperone Ssb on ribosomes.<br />(i) To investigate the functional interplay between the Ssb-RAC system and the nascent polypeptide-associated complex (NAC), another ribosome-anchored complex, genetic and biochemical approaches were applied. Deletion of the genes encoding Ssb resulted in the<br />accumulation of protein aggregates consisting predominantly of ribosomal proteins and<br />ribosome biogenesis factors. Additionally, the levels of ribosomal particles and actively translating ribosomes were reduced in these cells. These defects were aggravated in nacΔssbΔ cells, suggesting that both, Ssb and NAC, play a role in the regulation of ribosome biogenesis. The present work contributed the quantification of ribosome levels to this study,<br />as well as experiments showing that ribosomal proteins are specific components of the aggregates formed in ssbΔ and nacΔssbΔ cells.<br /><br /><br />(ii) To analyze the architecture of the RAC complex, which consists of Ssz and Zuotin,<br />pulldown experiments were performed showing that the N-terminal region of Zuotin is<br />sufficient to form a stable interaction with Ssz in vivo. This result complements the findings<br />obtained by amide hydrogen exchange experiments and mutational analyses. Together, the<br />data suggest that the mutual stabilization of the highly flexible N-terminus of Zuotin and the<br />C-terminal domain of Ssz constitutes the molecular basis for RAC complex formation.<br /><br /><br />(A) To investigate protein aggregation of disease-related Poly-Q (poly-glutamine) proteins, a<br />new method was developed for generating constructs containing repetitive sequences.<br />Cloning of repetitive DNA sequences using standard PCR-based methods is challenging due to the lack of specific primer binding sites. Therefore, a PCR-free seamless cloning strategy was designed to assemble highly repetitive nucleotide sequences. By this approach, DNA templates were generated to produce proteins containing defined stretches of consecutive glutamine residues in bacteria. With these proteins an improved assay was established to study the aggregation of Poly-Q polypeptides in vitro. Preißler, Steffen 2013-12-07T23:25:03Z Insights into cotranslational protein folding and protein quality control systems on ribosomes 2011-12-19T11:30:29Z Preißler, Steffen

Dateiabrufe seit 01.10.2014 (Informationen über die Zugriffsstatistik)

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