| Trypanosoma brucei spp. continue to impose serious socio-economic burdens throughout sub-Saharan Africa. Control of host infection is impeded by antigenic variation of the variant surface glycoprotein (VSG), a glycosylphosphatidylinositol (GPI) anchored homodimer, which covers the entire surface of the parasite. We have previously shown that the GPI anchor is a competent forward trafficking signal and is essential for VSG transport to the surface. The machinery that mediates this transit however, particularly through the early secretory pathway, remains uncharacterized in T. brucei and continues to be defined in other eukaryotes.;We show that the coat protein complex II (COPII) machinery, responsible for ER to Golgi trafficking, is conserved and functional in trypanosomes. Unlike other eukaryotes, trypanosomes express two paralogues each for the Sec23 and Sec24 subunits. All essential, they interact to form two obligate heterodimers, of which only one of is required for ER export of GPI-anchored cargo. Furthermore, these COPII components all co-localize to one or two ER exit sites, which nucleate at ER sub-domains associated with the flagellar attachment zone (FAZ). We suspect that this organization allows for the rapid transport of VSG through the cell, while also maintaining the fidelity of organellar segregation during cell division.;Concurrently, we have expanded our understanding of the trypanosome sphingolipid species. While these lipids do not affect VSG transport, their biosynthetic pathways have been targeted for the development of novel therapeutic drugs to control kinetoplastid infections. Relatively little is known about the sphingolipid synthases (SLSs) or the general sphingolipid composition in T. brucei. Using mass spectrometry and in vivo labeling studies we have more precisely defined the SL content in both stages of the parasite. T. brucei has four SLSs genes, and using a novel cell-free translation system, we have characterized their synthesis products and have identified key residues involved with their enzymatic specificity.;Collectively, this work delves into two fundamental topics in T. brucei: cargo transport through the early secretory pathway and sphingolipid synthesis. Though distantly related subjects, both projects have revealed novel (obligate COPII heterodimers) and unexpected (TbSLS biosynthetic diversity) details regarding the basic cell biology of this protozoan kinetoplastid. |
