Molecular cloning and characterization of a ras-related gene of ran/tc4/spi1 subfamily in Giardia lamblia.
The significance of Ras-like proteins in the protozoa is relatively unexplored. In this report, a gene encoding a Ras-like nuclear (Ran) protein was identified in Giardia lamblia by a polymerase chain reaction-based cloning strategy. The sequence analyses suggest that the gene was intronless, and had short 5'-untranslated leader sequences in the corresponding mRNA up to -2, -4, or -29 bases upstream of the first initiation codon. The full-length cDNA sequence predicted a protein comprising 226 amino acids, in which the highly conserved functional motifs of the Ras superfamily were all preserved. This protein showed 52% identity to human TC4 and 50% identity to yeast Spi1 proteins, suggesting that it is closely related to the Ran proteins, and it was therefore designated gRan. gRan produced from recombinant Escherichia coli exhibited GTP binding activity by an overlay assay. In good agreement with the predicted size of gRan, a 27-kDa protein was identified in a lysate of G. lamblia by Western blotting using antiserum raised against recombinant gRan. The protein was further localized in both nuclei of G. lamblia by immunofluorescence staining. Recombinant gRan exhibited low affinity for GTP with a Kd value of 16.8 microM. The affinity was enhanced to a Kd value of 2.2 microM in the presence of 10 mM Mg2+. The intrinsic GTPase activity of gRan was observed only in the presence of 10 mM Mg2+ and had an estimated Km of 5.6 microM and a Kcat of 0.33/h. These observations demonstrate the presence of Ras-like proteins in the most primitive eukaryotic cells, G. lamblia, and infer that the Ran protein may play a functional role in the nuclei of this organism.
Chen
L M
LM
Division of Infectious Diseases, Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, Republic of China.
Chern
Y
Y
Ong
S J
SJ
Tai
J H
JH
D000818:Animals; D001483:Base Sequence; D002451:Cell Compartmentation; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics*; D015870:Gene Expression; D017125:Genes, Protozoan; D011905:Genes, ras; D016829:Giardia lamblia / Q000235:genetics*; D006160:Guanosine Triphosphate / Q000378:metabolism; D007158:Immunologic Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D020931:ran GTP-Binding Protein
D017931:DNA Primers; D009687:Nuclear Proteins; D012333:RNA, Messenger; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020931:ran GTP-Binding Protein