TRYPANOSOMA-BRUCEI - CHARACTERIZATION OF A LIFE-CYCLE STAGE-SPECIFIC G-PROTEIN

G-proteins have important and diverse roles in cellular signaling in h igher eukaryotes but as yet little is known about the involvement of t hese GTP-binding proteins in protozoan parasites. Bacterial toxin-cata lysed ADP-ribosylation, routinely used in the characterisation of G-pr oteins in higher eukaryotes, was used to determine whether functional G-protein homologues could be detected in Trypanosama brucei. A 43-kDa polypeptide that was ADP-ribosylated in the presence of pertussis tox in was identified in membrane fractions. This polypeptide was also rec ognised by an antibody generated against a peptide sequence representi ng a mammalian G(O) alpha G-protein subunit. GTP and GTP gamma S (a no nhydrolysable analogue of GTP) abolished the ADP-ribosylation reaction , indicating that the 43-kDa polypeptide also binds GTP. Furthermore, this T. brucei G-protein is present in bloodstream but not procyclic l ife cycle stages. Using an in situ GTP binding assay, a further group of small-molecular-size GTP-binding proteins which are present in both bloodstream and procyclic stages have been identified. This demonstra tion of G-proteins in T. brucei suggests possible intracellular mechan isms that may be involved in the transduction of host or other parasit e signals. (C) 1995 Academic Press, Inc.