What do Pex4 and Pex22 do in apicomplexan membrane transport? - Project Summary Apicomplexan parasites have major impacts on human health e.g. Plasmodium falciparum causes malaria whereas Toxoplasma gondii causes opportunistic infections. The pathology of all apicomplexan-caused diseases resides in lytic replication cycles and/or the ensuing immune response. Lytic replication requires parasite invasion of an appropriate host cell, which is an essential step for their successful infection. Apicomplexa harbor a constellation of apically localized cytoskeletal and secretory organelles dedicated to host cell invasion. Biogenesis of and protein targeting to the three secretory organelles (micronemes, rhoptries, dense granules) relies on the secretory pathway, but so do other organelles like the apicoplast and the inner membrane complex (IMC). To correctly target vesicles and proteins to each of these destinations the parasite employs, like all eukaryotes, the ‘classic’ endoplasmic reticulum (ER) and Golgi apparatus, relying on Rab GTPases and SNARE proteins. However, in the diversified, latter half of trafficking, the Apicomplexa have repurposed aspects of the endosomal pathway, which is still mired with questions. In this proposal, the research team proposes to probe into the function of two peroxisome derived proteins, Pex4 and Pex22, that are universally conserved across Apicomplexa (most Apicomplexa completely lost all other peroxisome genes). Moreover, Pex4 and 22 are essential in the pathogenic stages of P. falciparum and T. gondii, while humans actually lost these two peroxisome genes. Pex4 is an E2 ubiquitin-conjugating (UBC) enzyme in complex with Pex22 that functions as a membrane anchor. In peroxisomes, they mediate cargo receptor recycling for the import of fully folded, co- factor bound proteins (complexes) from the cytoplasm. Clearly, this defines a unique set of proteins. However, the orthologous cargo receptor (Pex5) is missing in Apicomplexa and it is unclear on which organelle or set of proteins Pex4/22 act. The research team proposes to address these questions in T. gondii tachyzoites, for which powerful genetic and cell biological tools to dissect the secretory pathway are available. Presented preliminary data put Pex4/22 in the secretory way and depletion shows severe impacts on parasite fitness. The presented research plan will define the exact nature of where the Pex4/22 complex resides and how it acts. Conditional knock-downs will be paired with available compartment-specific markers to determine localization and functionality in the secretory pathway, while genetic complementation with mutant alleles will assess whether enzymatic activity and/or complex membrane anchoring is required. Furthermore, to identify the molecular context of the Pex4/22 process, TurboID complemented by organelle purification plus mass spectrometry will be performed. A sub-set of prioritized candidates will be experimentally validated to cement the nature and mechanism of the Pex4/22 complex in the apicomplexan secretory pathway. It is anticipated that Pex4/22 uniquely cater to an as yet unknown aspect of apicomplexan biology, and that the discoveries will write new chapters in both apicomplexan AND peroxisome biology.
