Structural characterisation of peroxisomal import receptor complexes

Peroxisomes are dynamic eukaryotic organelles that require import of their membrane and matrix proteins by soluble receptors in the cytosol. Their biogenesis, protein import, and proliferation are regulated by a distinct set of proteins, collectively called peroxins.Two peroxins, Pex3 and Pex19, are involved in the insertion of various Peroxisomal Membrane Proteins (PMPs) in the peroxisomal membrane and are thus crucial for its formation. Pex3 is a PMP that acts as a docking receptor for Pex19. The role of Pex19, in turn, is to bind, stabilise, and guide PMPs to the membrane-docked Pex3, where they are inserted into the peroxisomal membrane by an unknown mechanism. Pex14 is a PMP protein that is a key component of the peroxisomal import pore. Its interaction with Pex19 is established, yet little structural information is available about their full-length complex. In this thesis we investigated the conformation of the full-length human PEX19 in complex with the cytosolic domain of PEX3. A hybrid structural and biochemical approach was employed in order to characterize this interaction. Furthermore, the role of PEX19-PEX14 binding was addressed and the full-length complex was structurally characterised, providing insight into the stoichiometry, binding and shape of the never-before-described full-length assembly, using a variety of biochemical, biophysical and structural methods.The second part of this thesis aims to shed light on the peroxisomal protein import mechanism process. Matrix proteins (cargoes) can be imported into the peroxisomal lumen using peroxisomal targeting signal 1 (PTS1), or peroxisomal targeting signal 2 (PTS2) import pathways. The PTS1 pathway is the most common, utilising peroxin Pex5p. Pex5p is a soluble receptor, cycling between a free cytoplasmic state -where it recognises and binds peroxisomal matrix proteins- and a membrane bound state, as part of the transient PTS1 pore. The interaction between Pex5p and cargo proteins occurs via the TPR domains (tetratricopeptide repeats) of Pex5p and the PTS1 sequence at the C-terminus of the cargo proteins. Two peroxisomal cargoes, Pcs60 and MIF1, were studied in this part of the PhD project. Pcs60 is a yeast peroxisomal oxalyl-CoA synthetase, while MIF1 is a plant peroxisomal cargo associated with stress response, both of which contain a PTS1 recognition signal peptide. These proteins were crystallised and structurally characterised by means of X-ray crystallography. Further characterisation of Pcs60 and its complex with Pex5p was performed by biophysical methods, small angle X-ray scattering (SAXS), and negative stain electron microscopy. Elucidation of the structure of the Pex5p-Pcs60 complex will lead to enhancing our understanding of peroxisomal protein import