TcRho1, a farnesylated Rho family homologue from Trypanosoma cruzi

Rho GTPases are members of the Ras superfamily and are involved in signal t ransduction pathways, including maintenance of cell morphology and motility , cell cycle progression, and transcription activation. We report the molec ular identification in trypanosomatids (Trypanosoma cruzi) of the first mem ber of the Rho family. The cloned Rho protein, TcRho1, shares similar to 40 % homology with other members of the Rho family. Southern blot analysis rev ealed that TcRHO1 is a single copy gene per haploid genome, and Northern bl ot assays showed a transcript of 1200 nucleotides in length. Mapping the 5 ' -untranslated region of TcRHO1 transcripts revealed at least five differe nt transcripts derived from differential trans-splicing. Three of the five transcripts contain the trans-splicing site within the coding region of the TcRHO1 gene. TeRho1 also contains the C-terminal sequence CQLF (CAAX motif ), which is predicted to direct post-translation prenylation of the cystein e residue. A synthetic peptide containing this C-terminal motif, when teste d against Q-Sepharose chromatography fractions from T. cruzi cytosol, was s hown to be efficiently farnesylated, but not geranylgeranylated, despite th e fact that the CAAX motif with X = Phe specifies geranylgeranylation by ma mmalian protein geranylgeranyltransferase I. Furthermore, immunoblot analys es of epimastigote protein with anti-S-farnesyleysteine methyl ester and an ti-TcRhol antisera strongly suggested that TcRhol is farnesylated in vivo. The farnesylation of proteins such as Rho GTPases could be the basis for th e selective cytotoxic action of protein farnesyltransferase inhibitors on t rypanosomatids versus mammalian cells.