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Identifier 000434233
Title Single particle analysis of the peroxisomal protein importing machinery
Alternative Title Δομική ανάλυση του μοριακού μηχανισμού εισόδου πρωτεϊνών στα υπεροξειδοσώματα
Author Βίσμπας, Δημήτριος Θ.
Thesis advisor Gatsogiannis, Christos
Κοκκινίδης, Μιχαήλ
Reviewer Αθανασάκη, Ειρήνη
Πετράτος, Κυριάκος
Abstract Pex14p, Pex17p and Pex5p peroxins, are the key components of the protein import machinery of Saccharomyces cerevisiae from the cytosol to the peroxisomal lumen. It is suggested that these peroxins are sufficient for the assembly of a functional importomer complex. However, the importing mechanism still remains unclear. The field is lacking structural insights into higher order membrane assemblies and markedly, into the pore formation mechanism. In the framework of this thesis, the yeast (Pex14p, Pex14p/Pex17p) and human (PEX14) docking complex components were reconstituted into lipid nanodiscs and further optimized for electron microscopy (EM) analysis, in order to elucidate the Pex17p contribution to the docking complex and reveal differences between the yeast and human homologues. Additionally, reconstitution of the yeast docking complex Pex14p/Pex17p in liposomes and subsequent addition of Pex5p was performed in order to clarify the structure and topology of the importomer. Initially, negative stain EM comparative studies between the Pex14p/Pex17p complex and Pex14p in absence of Pex17p suggest that Pex17p provides stability to the docking complex. Following, the human homologue PEX14 was expressed, purified and successfully reconstituted into lipid nanodiscs. Characterization with negative stain EM was performed but extensive optimization of the vitrification conditions is required in order to proceed with cryo-EM studies and observe substantial differences between the two homologues. Finally, liposomes containing the yeast docking complex (Pex14p.Pex17p) were assembled successfully. However, negative stain analysis after addition of Pex5p did not reveal any structural changes on the liposome surface. A close to native environment analysis, using cryo-EM, will be required for a more detailed data acquisition. Overall, the results of this thesis, set the stage for future pore-formation in vitro studies, using cargo proteins.
Language English
Subject Cryo-EM
Import membranes
Εισαγωγή μεμβράνης
Issue date 2020-11-27
Collection   School/Department--School of Sciences and Engineering--Department of Biology--Post-graduate theses
  Type of Work--Post-graduate theses
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