A Tale of Two Barrels: Import of β-Barrel Proteins into Yeast Mitochondria

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dc.contributor.advisor Rapaport, Doron (Prof. Dr.)
dc.contributor.author Moitra, Anasuya
dc.date.accessioned 2023-01-30T14:50:37Z
dc.date.available 2023-01-30T14:50:37Z
dc.date.issued 2023-01-30
dc.identifier.uri http://hdl.handle.net/10900/135827
dc.identifier.uri http://nbn-resolving.de/urn:nbn:de:bsz:21-dspace-1358275 de_DE
dc.identifier.uri http://dx.doi.org/10.15496/publikation-77178
dc.description.abstract Membrane-embedded β-barrel proteins are found in the outer membranes (OM) of Gram-negative bacteria, mitochondria and chloroplasts. They form a barrel shaped hydrophilic pore in the membrane, which can be composed of 8-26 H-bonded, mostly anti-parallel β-strands. These proteins constitute an essential part of the OM proteome by functioning as transporters, enzymes, or subunits of protein translocons and membrane insertion machineries. Mitochondrial and chloroplast β-barrel proteins are transcribed in the nucleus and translated on cytosolic ribosomes, from where they are targeted to the correct subcellular organelle and ultimately integrated with the help of dedicated import machineries into the respective outer membrane. The biogenesis pathways for β-barrel proteins have been quite conserved over the course of evolution due to the endosymbiotic origins of mitochondria and chloroplasts from ancient Gram-negative bacterial endosymbionts (α-proteobacterium and a photosynthetic cyanobacterium, respectively). Such conserved mechanisms prompted studies to investigate the evolutionary lineage of these pathways. Bacterial and chloroplast β-barrel proteins have been shown to be targeted and integrated into mitochondria upon their expression in yeast cells. In this study, I aimed to study the extent of evolutionary conservation of β-barrel biogenesis pathways. To this end, I started my work by testing the destiny of the bacterial secretins InvG and SsaC upon their expression in yeast cells. My findings demonstrate that they are capable of mitochondrial localization in these cells and their biogenesis is variably dependent on the TOM import receptors and the TOB complex. Next, I investigated whether de novo designed synthetic eight-stranded transmembrane β-barrel (TMB) proteins (Tmb2.3 and Tmb2.17) could be expressed in yeast cells and integrated into their mitochondria. I further assessed the role of components of yeast mitochondrial import and assembly machinery in their proper biogenesis. My results demonstrate that both de novo designed synthetic TMBs, Tmb2.3 and Tmb2.17, can be successfully expressed as HA-tagged proteins in yeast cells without being detrimental to their growth. Subcellular fractionation experiments show that both TMBs can be targeted to the mitochondria, with partial ER localization as well. They are embedded into the mitochondrial OM with a topology that exposes part of them to the cytosol. I further observed that the absence of either one of the import receptors (Tom20 or Tom70), did not impair their biogenesis. Moreover, deficiency in certain critical components of the TOB complex, namely Mas37 and Tob55, differentially affected the assembly of these synthetic TMBs. In the absence of Mas37, the mitochondrial steady state levels of the TMBs decreases, similar to bona fide mitochondrial β-barrel proteins. Depletion of Tob55 leads to decreased mitochondrial levels of Tmb2.3-HA, while the levels of Tmb2.17-HA are further stabilized. Interestingly, Tmb2.3-HA can assemble into higher oligomers in a TOB complex dependent manner. In contrast, the biogenesis of Tmb2.17-HA seems to be independent of the TOB complex. Collectively, my findings indicate that different β-barrel proteins can be dependent to a variable extent on the proper function of the TOB complex. Such distinctions might be mapped to the dissimilarities in their sequence, hydrophobicity patterns of the β-strands, and/or different sequence of the classical β-signal at the last β-strand. My results further suggest the strong evolutionary conservation of pathways and machineries that recognize β-barrel structural elements and facilitate the biogenesis of β-barrel proteins across the spectrum of life. en
dc.language.iso en de_DE
dc.publisher Universität Tübingen de_DE
dc.rights ubt-podok de_DE
dc.rights.uri http://tobias-lib.uni-tuebingen.de/doku/lic_mit_pod.php?la=de de_DE
dc.rights.uri http://tobias-lib.uni-tuebingen.de/doku/lic_mit_pod.php?la=en en
dc.subject.ddc 570 de_DE
dc.subject.other β-barrel proteins en
dc.subject.other de novo designed proteins en
dc.subject.other mitochondria en
dc.subject.other outer membrane en
dc.subject.other TOB complex en
dc.subject.other yeast en
dc.title A Tale of Two Barrels: Import of β-Barrel Proteins into Yeast Mitochondria en
dc.type PhDThesis de_DE
dcterms.dateAccepted 2023-01-10
utue.publikation.fachbereich Biochemie de_DE
utue.publikation.fakultaet 7 Mathematisch-Naturwissenschaftliche Fakultät de_DE
utue.publikation.noppn yes de_DE


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