Network analysis of co-translationally acting factors mediating membrane targeting of proteins in E. coli
Metabolism, Biochemistry and Genetics of Microorganisms
Final Report Abstract
The major goal of the proposal was to understand the role of protein maturation factors in the cotranslational triaging and maturation of nascent polypeptides; the interplay of factors at the ribosome, and the extent of antagonistic or collaborative activities that contribute to the robustness of the system. We reveal that TF orchestrates the nascent chain engagement of all factors acting downstream in folding and translocation, including SecB, DnaK and GroEL. We describe the multiple roles of SecB in protein maturation, ranging from the support of CP folding to the support of IMP membrane insertion and we collaborated with the Mraheil laboratory to reveal the ribosome interaction of the SecA2 from Listeria monocytogenes. Finally, we provide indications that the chaperone DnaK is implicated in IMP biogenesis by supporting the folding of cytosolic domains. The study reveals that chaperones are integrated components of both translocation pathways by contributing to substrate triaging, membrane insertion and the folding of cytosolic domains. In the course of this project, we developed new methodology, including (i) a new, statistically sound data analysis and heuristics for interpreting and comparing SeRP data; (ii) a new SeRP protocol to identify membrane-engaged ribosomes and (iii) a spatial proteomics screen to quantitatively assesses the proteome distribution among different compartments in E. coli. Analyzing the SecA function by SeRP was highly challenging and the project was eventually stopped before completion. Establishing a SecA SeRP protocol was unexpectedly difficult and required multiple attempts. At the end of the development process, we were informed by Prof. Dr. Shu-ou Shan (Caltech, California) that her lab had performed a SeRP study that revealed the SecA nascent interactome in wildtype cells and in mutants lacking SecB and TF. In response to this, we shifted our focus on the analysis of the role of chaperones TF, the Hsp70 DnaK and the Hsp60 GroEL in cotranslational folding and targeting. This study is now completed and revealed, among others, a new role of DnaK in IMP biogenesis, as described above. The analysis of the impact of translation speed on translocation is ongoing (see below) and requires further ribosome profiling experiments and the adaptation of available data analyses tools, to also incorporate information of 60 nt ribosome footprints that reveal the position of collided disomes, another recently established indicator for translation pausing. The progress of the project was strongly delayed by two different incidents: First, the lab was closed in response to the Corona pandemic. Second, lab work was stopped for several months in response to a closure of the ZMBH due to a waterpipe break that destroyed the electric system of the building.
Publications
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Selective ribosome profiling to study interactions of translating ribosomes in yeast. Nature Protocols, 14(8), 2279-2317.
Galmozzi, Carla V.; Merker, Dorina; Friedrich, Ulrike A.; Döring, Kristina & Kramer, Günter
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Mechanisms of Cotranslational Protein Maturation in Bacteria. Frontiers in Molecular Biosciences, 8 (2021, 5, 25).
Koubek, Jiří; Schmitt, Jaro; Galmozzi, Carla Veronica & Kramer, Günter
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SecA2 Associates with Translating Ribosomes and Contributes to the Secretion of Potent IFN-β Inducing RNAs. International Journal of Molecular Sciences, 23(23), 15021.
Teubner, Lisa; Frantz, Renate; La, Pietra Luigi; Hudel, Martina; Bazant, Jasmin; Lochnit, Günter; Eismann, Lena; Kramer, Günter; Chakraborty, Trinad & Abu, Mraheil Mobarak
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Selective ribosome profiling reveals a role for SecB in the co-translational inner membrane protein biogenesis. Cell Reports, 41(10), 111776.
Eismann, Lena; Fijalkowski, Igor; Galmozzi, Carla Verónica; Koubek, Jiří; Tippmann, Frank; Van, Damme Petra & Kramer, Günter
