Peroxisomes are membrane-bound subcellular compartments found in virtually all eukaryotic cells. These evolutionary conserved organelles are indispensable in homeostasis and disease. Peroxisomes have versatile functions in different organisms and tissues. Peroxisomes are involved in fatty acid oxidation (mammals), glyoxylate cycle (plants and yeasts), photorespiration and hormone biosynthesis (plants), glycolysis (protists) and many other biological functions.

Peroxisomes lack protein synthetic abilities and therefore their function is dependent on protein import. Commonly, the cell organelles import proteins synthesized on the ER using vesicle mediated routes. Peroxisomes are distinguished in this regard, since the majority of peroxisomal proteins are synthesized on free-ribosomes in the cytoplasm and imported by direct transmembrane transport catalysed by the PEX translocon. The translocon is composed of at least two dozens proteins known as peroxins which recognize the protein to be imported (hereafter cargo) in the cytoplasm, dock the recognition complex at the surface of the organelle, translocate the cargo and regenerate the system for further import cycles. Most of the peroxins have been identified by genetic studies by observing the effects of mutations and deficiencies in human disease and in yeast. The structural organization of the PEX translocon and mechanistic functions of most of the peroxins remain incompletely understood. The major objective of this proposal is in structural and functional characterization of the key aspects of peroxisomal protein import: recognition, docking and translocation in human and protists.

Cargos, directed to the peroxisome are distinguished by virtue of Peroxisomal Targeting Sequences of type 1 (PTS1) or type 2 (PTS2). These short stretches of amino-acids are recognized in the cytoplasm by PEX5 or PEX5/PEX7, respectively. The recognition complex then docks at PEX14 and PEX13 - receptors integral to the peroxisomal membrane. Translocation is supported by the latter receptors, and possibly by other peroxins, but the process itself remains almost an unknown. In this proposal, using protein crystallography,single particle cryo-electron microscopy (cryo-EM) and cryotomography (cryo-ET) we will cast light on the structural organization of these exciting processes.