Analysis of CDPK1 targets identifies a trafficking adaptor complex that regulates microneme exocytosis in Toxoplasma
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chan-awchan-phd-biology-2023-thesis.pdf
Description
Thesis PDF
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7.82 MB
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Adobe PDF
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8a91321fa548502018e8740fe1c24e32
Author(s)
Chan, Alex Wai
Advisor(s)
Lourido, Sebastian
Date Issued
June 2023
Publisher
Massachusetts Institute of Technology
Abstract
Apicomplexan parasites use Ca²⁺-regulated exocytosis to secrete essential virulence factors from specialized organelles called micronemes. Ca²⁺-dependent protein kinases (CDPKs) are required for microneme exocytosis; however, the molecular events that regulate trafficking and fusion of micronemes with the plasma membrane remain unresolved.
In this thesis, I describe the discovery and characterization of a regulator of microneme exocytosis in Toxoplasma gondii. In the first chapter, I introduce T. gondii as a model apicomplexan to study motile stages during asexual stages. In the second chapter, I discuss combining sub-minute resolution phosphoproteomics and bio-orthogonal labeling of kinase substrates in T. gondii to identify 163 proteins phosphorylated in a CDPK1-dependent manner. In addition to known regulators of secretion, I identify uncharacterized targets with predicted functions across signaling, gene expression, trafficking, metabolism, and ion homeostasis. In the third chapter, I describe the functional characterization of a target of CDPK1, the putative activating adaptor HOOK. In other eukaryotes, HOOK homologs form the FHF complex with FTS and FHIP to activate dynein-mediated trafficking of endosomes along microtubules. I show the FHF complex is partially conserved in T. gondii, consisting of HOOK, an FTS homolog, and two parasite-specific proteins (TGGT1_306920 and TGGT1_316650). CDPK1 kinase activity and HOOK are required for the rapid apical trafficking of micronemes as parasites initiate motility. Moreover, parasites lacking HOOK or FTS display impaired secretion of microneme proteins, leading to a block in the invasion of host cells. Taken together, our work provides a comprehensive catalog of CDPK1 targets and reveals how vesicular trafficking has been tuned to support a parasitic lifestyle.
MIT Department
Massachusetts Institute of Technology. Department of Biology
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