Despite being recognized as an important organelle in the secretory pathway, not many aspects of endoplasmic reticulum (ER) protein import are understood in Trypanosoma brucei, the causative agent of human African trypanosomiasis (HAT). Movement of proteins into the ER requires a signal sequence and a Sec61p translocation pore. We have developed a cell-free system using T. brucei microsomes (TbRM) and variant surface glycoprotein (VSG) as our model substrate. TbRM could import full-length proteins post-translationally in a parasitecytosol dependent fashion. Purified Hsp70 (Ssa1p) could replace parasite cytosol in assisting protein translocation into TbRM, suggesting a role of molecular chaperones in protein translocation into ER of trypanosomes. Protein import into TbRM was signal peptide dependent. Further, model proteins from Escherichia coli, Saccharomyces cerevisiae and Bos taurus were not imported into TbRM. Conversely, many trypanosomatid proteins were not imported into canine microsomes. Since signal peptides are selectively recognized by specific translocation machineries, we hypothesize that signal peptides have co-evolved with their translocons.We used the in vitro system to identify protein translocation blockers (PTBs), compounds that inhibit parasite protein import into TbRM. PTBs (MAL3-101, equisetin and CJ-21, 058) were trypanocidal, with IC50 (the concentration at which fifty percent of parasites are killed) of; 125 nM (MAL3-101), 3.3 M (equisetin) and 7 M (CJ-21, 058). PTBs did not affect viability of a model mammalian cell. Therefore, we have i) identified ER protein translocation machinery, as a drug target in T. brucei, and ii) discovered trypanocidal compounds that may act on import of proteins into the parasite ER. Signal sequences have three conserved regions; an amino-terminal (n-region), a hydrophobic (h-region), and a carboxy-terminal (c-region). Although the hydrophobic core is important for efficient import/export of proteins, functional characteristics of h-regions are poorly understood. A bioinformatics analysis led to the discovery of highly conserved peptide motifs in h-region of signal sequences in eukaryotes (h. sapiens, S. cerevisiae, T. brucei), and prokaryotes (E. coli, B. subtilis). Peptide motifs in h-regions may significantly contribute to signal sequence activity. Finally, amino acids in h-region of signal sequences are not randomly selected. On the contrary, selection and distribution of amino acids in h-regions is highly specific to, and varies across species.
