Biochemical and genetic evidence for a family of heterotrimeric G-proteins in Trichomonas vaginalis.
We have cloned a single copy gene from the human parasite Trichomonas vaginalis that encodes a putative protein of 402 amino acids with approximately 35% sequence identity to known alpha subunits of heterotrimeric G-proteins. It contains the characteristic GTP binding domains G-1 to G-5 with the key residues conserved. The new sequence has an unusual N-terminal extension of approximately 70 residues that cannot be aligned to reference G-proteins and which is characterised by proline-rich repeats. To investigate the expression and cellular localisation of the protein we produced specific antisera against a recombinant fusion protein. The antisera recognised a protein of an apparent molecular mass of 51 kDa in protein extracts from T. vaginalis and immunofluorescent microscopy established that the protein is localised to discrete endomembranes. Using a protocol designed to purify mammalian heterotrimeric G-proteins incorporating a GTPgammaS binding assay, we isolated two proteins from Trichomonas that are recognised by an heterologous GA/1 antisera raised to a peptide of the conserved G-1 domain of G-protein alpha subunits. These two proteins have an apparent molecular mass of 61 and 48 kDa, respectively, larger and smaller than the translation product of the cloned gene. Consistent with these results, the GA/1 antisera did not cross-react with the fusion protein produced from the gene we have cloned. These data suggest T. vaginalis possesses more than one heterotrimeric G-protein alpha subunit. Based on the sequence features of the cloned gene and the biochemical properties of the purified proteins, we suggest that these alpha subunits are likely to be part of classic heterotrimeric G-protein complexes.
Hirt
R P
RP
Department of Zoology, The Natural History Museum, Cromwell Rd, London SW7 5BD, UK. rch@nhm.ac.uk
Lal
K
K
Pinxteren
J
J
Warwicker
J
J
Healy
B
B
Coombs
G H
GH
Field
M C
MC
Embley
T M
TM
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D017124:Conserved Sequence; D017125:Genes, Protozoan; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D020449:Repetitive Sequences, Amino Acid; D016415:Sequence Alignment; D014246:Trichomonas vaginalis / Q000737:chemistry* / Q000235:genetics
C476843:Galpha402 protein, Trichomonas vaginalis; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D020962:Heterotrimeric GTP-Binding Proteins