ENDOPLASMIC RETICULUM OF TRYPANOSOMATIDS

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DESCRIPTION (provided by the applicant): Trypanosomatid surface proteins (e.g., variant surface glycoprotein, VSG of Trypanosoma brucei) are critical for establishment of infections. Import into the endoplasmic reticulum (ER) is the committed step or entry into the exocytic pathway for these polypeptides. Hence, ER protein translocation is a very important pathway in the parasite. Our long-term objective is to characterize the trypanosomatid ER import machinery, as most fundamental properties of the ER protein import have not been established. Translocation of proteins into the ER of eukaryotes is dependent on an N-terminal signal sequence, signal recognition particle (SRP), SRP receptor (5R), and a translocon (formed by the 5ec61 p complex). Protein import takes place either co-translationally or post-translationally, and it is widely held that signal peptides are interchangeable across all biological families We have found species-specificity in signal sequence recognition: signal peptides from T. brucei and Leishmania do not operate efficiently (if at all) with vertebrate microsomal import machineries. Our immediate goal is to develop an in vitro system that is capable of importing proteins from trypanosomes, since the canine microsomal system is worthless for this purpose. To this end, membranes from T. brucei will be fractionated, and optimized for import of VSG. Availability of a T. brucei microsomal system will facilitate characterization of (i) properties of trypanosome signal peptides, (ii) delineation of the biochemical properties of the T. brucei translocation system, and (iii) identification of molecular differences between host and T. brucei translocation machineries. Understanding the molecular basis of the species-specificity of signal peptide selection between a trypanosome and a mammalian host is important, because the information may provide leads for rationale development of anti-trypanosome drugs.