Cloning and analysis of PTS-1 receptor in Trypanosoma brucei

Kinetoplastid organisms, such as the protozoan parasite Trypanosoma brucei, compartmentalise several important metabolic pathways in organelles called glycosomes. Glycosomes are related to peroxisomes of yeast and mammalian c ells. A subset of glycosomal matrix proteins is routed to the organelles vi a the peroxisome-targeting signal type 1 (PTS-1). The PEX5 gene homologue h as been cloned from T. brucei coding for a protein of the translocation mac hinery, the PTS-1 receptor. The gene code for a polypeptide of 654 amino ac ids with a calculated molecular mass of 70 kDa. Like its homologue in other organisms T. brucei PTS-1 receptor protein (TbPEX5) is a member of the tet ratricopeptide repeat (TPR) protein family and contains several copies of t he pentapeptide W-X-X-X-F/Y. Northern and Western blot analysis showed that the protein is expressed at different stages of the life cycle of the para site. The protein has been overproduced in Escherichia coli and purified us ing immobilized metal affinity chromatography. The purified protein specifi cally interacts in vitro with glycosomal phosphoglycerate kinase-C (PGK-C) of T. brucei, a PTS-1 containing protein. The equilibrium dissociation cons tant (K-d) of PGK-C for purified TbPEX5 is 40 nM. Using biochemical and cyt ochemical techniques a predominately cytosolic localization was found for T bPEX5. This is consistent with the idea of receptor cycling between the gly cosomes and the cytosol. (C) 1999 Elsevier Science B.V. All rights reserved .