Molecular mechanisms of protein trafficking to the lysosome in Trypanosoma bruce

Trypanosoma brucei spp are protozoan parasites that cause human and veterinary trypanosomiasis in sub-Saharan Africa. Current trypanocidal compounds used to combat these diseases are inadequate, suggesting the need for the development of better chemotherapeutic compounds. The lysosome in these parasites is critical for their sustenance within their respective ecological niches. Studies on the mechanisms involved in proper lysosomal biogenesis and function could therefore be very helpful in the development of these new therapies. Such studies can also shed more light on eukaryotic trafficking mechanisms that have been conserved through millions of years of evolution.;The molecular mechanism of trafficking of lysosome resident proteins in T. brucei is not well understood. We have previously shown that the cytoplasmic domain (CD) of the lysosome integral membrane glycoprotein p67 is both necessary and sufficient for trafficking in procyclic trypanosomes. The specific residues within this domain that are critical for this process are, however, not known. Present within this domain are two amino acid motifs that conform to the canonical [DE]XXXL[LI] acidic dileucine motif sequence that have been shown to mediate the trafficking of various transmembrane proteins to the lysosome in higher eukaryotes. Using site-specific mutagenesis and a GFP:p67CD fusion reporter system, this study shows that these dileucine motifs are critical in mediating trafficking to the lysosome in procyclic trypanosomes. Though both motifs are needed for maximal trafficking, the membrane distal motif plays a dominant role in this process. Additional studies implicate the acidic residues located upstream of the dileucine motifs in mediating the exit of p67 from the endoplasmic reticulum.;In higher eukaryotes, lysosome resident proteins are sorted into specific clathrin-coated vesicles for trafficking. The existence and utilization of such vesicles in T. brucei is currently an open question. This study further implicates a putative homologue of the medium subunit of the adaptor protein 1 complex in T. brucei in mediating the trafficking of both integral membrane and soluble residents of the lysosome.;Overall, the work presented in this dissertation explores some of the molecular aspects of the signal-directed mechanisms that ensure the efficient and effective trafficking of lysosomal proteins in Trypanosoma brucei .