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J. Biol. Chem., Vol. 276, Issue 32, 29711-29718, August 10, 2001
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From the Rho GTPases are members of the Ras superfamily
and are involved in signal transduction pathways, including maintenance
of cell morphology and motility, cell cycle progression, and
transcription activation. We report the molecular identification in
trypanosomatids (Trypanosoma cruzi) of the first member of
the Rho family. The cloned Rho protein, TcRho1, shares ~40% homology
with other members of the Rho family. Southern blot analysis revealed
that TcRHO1 is a single copy gene per haploid
genome, and Northern blot assays showed a transcript of 1200 nucleotides in length. Mapping the 5'-untranslated region of
TcRHO1 transcripts revealed at least five different
transcripts derived from differential trans-splicing. Three of the five
transcripts contain the trans-splicing site within the coding region of
the TcRHO1 gene. TcRho1 also contains the C-terminal
sequence CQLF (CAAX motif), which is predicted to
direct post-translation prenylation of the cysteine residue. A
synthetic peptide containing this C-terminal motif, when tested against
Q-Sepharose chromatography fractions from T. cruzi cytosol, was shown to be efficiently farnesylated, but not geranylgeranylated, despite the fact that the CAAX motif with
X = Phe specifies geranylgeranylation by mammalian
protein geranylgeranyltransferase I. Furthermore, immunoblot analyses
of epimastigote protein with anti-S-farnesylcysteine methyl
ester and anti-TcRho1 antisera strongly suggested that TcRho1 is
farnesylated in vivo. The farnesylation of proteins such as
Rho GTPases could be the basis for the selective cytotoxic action of
protein farnesyltransferase inhibitors on trypanosomatids versus mammalian cells.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF177587.
TcRho1, a Farnesylated Rho Family Homologue from
Trypanosoma cruzi
CLONING, TRANS-SPLICING, AND PRENYLATION STUDIES*
§¶
,,,,,,,¶§§, and¶¶
Instituto de Biofísica Carlos
Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro
21949, Brazil, the Departments of § Chemistry,
¶ Biochemistry, and Medicine,
University of Washington, Seattle, Washington 98195, and
** INSERM U397, Institut C. Regaud 20-24 rue du pont
Saint-Pierre, 31052 Toulouse Cedex, France
*
This work was supported by National Institutes of Health
Grant CA52874 (to M. H. G.) and Grant 661030/1996-2 from the Programa de Nucleos de Excelencia/Conselho Nacional de Desenvolvimento Cientifico e Technologico (PRONEX/CNPq).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Both authors contributed equally to this work.
§§
To whom correspondence may be addressed: Depts. of Chemistry and
Biochemistry, University of Washington, Seattle, WA 98195. Tel.:
206-543-7142; Fax: 206-685-8665; E-mail: gelb@chem.washington.edu.
¶¶
To whom correspondence may be addressed: Lab. de
Parasitologia Molecular, IBCCF, Universidade Federal do Rio de Janeiro,
CCS, Cidade Universitária, Rio de Janeiro 21949, Brazil. Tel.:
55-21-012-562-6540; Fax: 55-21-280-8193; E-mail:
lopesu@biof.ufrj.br.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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