Cellular and Molecular Determinants of Flagellar Membrane Localization of the Trypanosoma cruzi Flagellar Calcium-binding Protein

Trypanosomes are flagellated unicellular eukaryotic parasites that are responsible for tremendous morbidity and mortality worldwide. The studies described herein focus primarily on Trypanosoma cruzi, the etiologic agent of Chagas disease, a debilitating and potentially life threatening condition that is prevalent throughout Mexico, Central and South America. Besides being of interest for their global health burden, trypanosomes serve as excellent model organisms for the elucidation of mechanisms underlying ciliary trafficking. The plasma membrane of trypanosomes contains three distinct membranous domains that include cell body, flagellar and flagellar pocket membranes. While no physical barrier separates the membranes of these contiguous structures, the flagellar membrane is both structurally and functionally distinct from the flagellar pocket and pellicular membranes, and it has unique protein and lipid composition. There is an apparent asymmetric distribution of certain proteins across these membrane domains, and several proteins have been identified across trypanosomes that are heavily enriched or restricted to the flagellar membrane. The flagellar membrane of trypanosomes is enriched in lipid raft components, which might serve to recruit and/or retain certain types of flagellar membrane proteins.;The flagellar calcium binding protein (FCaBP) of T. cruzi is localized to the flagellar membrane in all life cycle stages of the parasite. The myristoylated and palmitoylated N-terminus of FCaBP is necessary and sufficient for the flagellar membrane targeting. Not all dually acylated proteins are flagellar, however. I generated T. cruzi transfectants expressing the N-terminal 24 or 12 amino acids of FCaBP fused to GFP. Analysis of these mutants revealed that while amino acids 1-12 are sufficient for dual acylation and membrane binding, amino acids 13-24 are required for flagellar specificity and lipid raft association. Mutagenesis of several conserved lysines in the latter peptide revealed that these residues are essential for flagellar targeting and lipid raft association. These lysine residues may promote electrostatic interactions with negatively charged head groups on the inner leaflet of the flagellar membrane. While a similar combination of acylation and electrostatic membrane interaction has been observed in mammals, this is the first described example of such interaction in a primitive eukaryote.;Considered together with the work of others, my results indicate that the flagellar membrane localization of some proteins requires an association with lipid raft microdomains. For FCaBP, several elements---myristoylation, palmitoylation and a cluster of nearby basic amino acids---are each required to collectively support this protein-lipid interaction. The failure of other dually-acylated proteins to localize to the flagellar membrane can thus be explained by the insufficiency of dual acylation alone to target lipid rafts. Alternatively, a protein containing additional domains that interact with another cellular protein might possibly override flagellar membrane targeting.;FCaBP undergoes substantial change in conformation upon binding of calcium, reminiscent of recoverin. Unlike in Rv, the acyl groups of FCaBP are exposed regardless of calcium levels, so that both the calcium free and calcium bound forms of FCaBP may be anchored to the flagellar membrane. Also, unlike Rv FCaBP forms dimers in solution independent of calcium and the role of dimerization in function of FCaBP remains yet to be determined.;In addition to identifying the common domains contributing to flagellar or ciliary membrane association, a dissection of the determinants for protein and lipid sorting from the Golgi to target organelles will be valuable. Understanding the basis for the asymmetric distribution of proteins and lipids across cellular membranes is a fundamental step towards understanding cell polarization. Such polarization, whether of epithelial or endothelial barriers, immune cells, neurons, and possibly host cells undergoing invasion by pathogens, is a hallmark of complex organisms. Unraveling these molecular mechanisms for protein trafficking, localization and function in protozoans may contribute greatly to our understanding of human biology.