Insight into the Intermolecular Interactions and Functionality of the Recombinant PEX19-PEX26 Complex

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dc.contributor.advisor Dodt, Gabriele (Prof. Dr.)
dc.contributor.author Koçak, Erman
dc.date.accessioned 2019-08-29T09:13:44Z
dc.date.available 2019-08-29T09:13:44Z
dc.date.issued 2021-08-22
dc.identifier.uri http://hdl.handle.net/10900/92155
dc.identifier.uri http://nbn-resolving.de/urn:nbn:de:bsz:21-dspace-921554 de_DE
dc.identifier.uri http://dx.doi.org/10.15496/publikation-33536
dc.description.abstract Peroxisomal membrane proteins (PMPs) function in various mode of actions as membrane receptors or docking proteins. They are mainly employed in the import of metabolites or matrix proteins as well as in the export machinery of the matrix protein receptor PEX5. PEX3 as one of class II PMPs serves as a docking protein on the peroxisome membrane for PEX19. Lack of PEX3 or PEX19 results in the absence of peroxisomes, as well as of peroxisomal remnants and in the degradation or mislocalisation of PMPs to other organelles such as mitochondria. PEX19 is a chaperone-like receptor protein recruiting class I PMPs that are employed in transport of metabolites like PMP22, PMP34 and PMP70 or are components of the docking-translocation complex (DTM) like PEX2, PEX10, PEX12 and PEX13. Besides that, PEX19 is also essential for the PEX26 import into the peroxisomal membrane, a protein that is one of the components of the receptor export module (REM). PEX26 as one of these tail-anchored (TA) class I PMPs is essential for the export of PEX5, recruiting the AAA-ATPase (PEX1-PEX6) to the peroxisomal membrane forming the REM. Depletion of the AAA-ATPase in HeLa cells results in accumulation of monoubiquitinated PEX5, thus in impaired matrix protein import. Phenotypically, deficiency of PEX26 was reported in all CG8-patients (Complementation Group 8) suffering from Zellweger syndrome which is referred to as the most severe peroxisomal biogenesis disorder. Overexpression of PEX26-cDNA in E. coli resulted in highly insoluble or aggregated PEX26. In contrast, its co-expression with PEX19 revealed a stable and soluble binary complex. Here we could successfully overexpress PEX19 and PEX26 as a binary complex through a two-promoter E. coli system without using any denaturing agent for the solubilisation. This provided us with an in vitro native-like complex that could be analysed by using two different methods, size exclusion chromatography and native-MS. Furthermore, we elucidated the interacting domains of this binary complex by XL-MS employing the cross-linking reagent BS3. This revealed the proximity of the domains that are able to interact with each other in the native-like complex of PEX19-PEX26. Regarding functionality, we showed that this binary complex was able to restore matrix protein import in vivo in PEX26-deficient fibroblasts re-establishing the REM. Last but not least, we successfully integrated PEX26 into liposomal membranes employing Ni-NTA-LUVs (large unilamellar vesicles) which were previously coupled with nPEX3T (Thx-His6-Thr-TEV-nPEX3). Following this, flow cytometry analysis revealed that PEX26 can be effectively integrated into these LUVs by nPEX3T. The membrane association of PEX26 could be tested using carbonate extraction, which confirmed PEX26 integration into LUVs. 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.classification Biochemie , Proteine , Peroxisom , Interaktion , Funktion , Struktur de_DE
dc.subject.ddc 500 de_DE
dc.subject.ddc 610 de_DE
dc.title Insight into the Intermolecular Interactions and Functionality of the Recombinant PEX19-PEX26 Complex en
dc.type PhDThesis de_DE
dcterms.dateAccepted 2019-08-22
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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