The Function of Phosphatidylinositol 4-Kinase III-Beta in Trypanosoma Brucei




Rodgers, Melissa Jeane

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Phosphatidylinositol 4-kinase phosphorylates phosphatidylinositol at the D4 position resulting in phosphatidylinositol 4-monophosphate. Subsequent phosphorylation events result in a group of molecules known as phosphoinositides. These molecules are important in signal transduction, endocytosis, exocytosis, and protein trafficking. Two phosphatidylinositol 4-kinases are found in Trypanosoma Brucei. We have cloned the gene encoding the Type III phosphatidylinositol 4-kinase Beta (TbPI4KIII-ß) in Trypanosoma Brucei. The protein was exogenously expressed in COS-7 cells and assayed for phosphatidylinositol kinase activity. The expressed protein migrated on SDS-PAGE near the predicted molecular weight of 66 kDa and phosphorylates phosphatidylinositol. Depletion of TbPI4KIII-ß in procyclic T. brucei by RNAi results in inhibition of cell growth and a distorted cellular morphology. Immunofluorescence studies revealed a distorted Golgi apparatus and mislocalization of lysosomal and flagellar pocket proteins. Ultrastructural analysis reveals internal accumulation of a heterogeneous population of vesicles, abnormal positioning of organelles and a loss of cell polarity. Scanning EM reveals a twisted morphology most likely due to an alteration in the microtubule cytoskeleton. Dividing TbPI4KIII-ß RNAi trypanosomes often exhibited a detached daughter flagellum and lacked a cleavage furrow, suggesting a defect in cell division and/or cytokinesis. Cell cycle analysis confirmed that cells depleted of TbPI4KIII-ß have a post-mitotic cytokinesis block. In summary, TbPI4KIII-ß is an essential protein in procyclic T. brucei, required for maintenance of Golgi structure, protein trafficking, normal cellular shape and cytokinesis.

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