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.
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