Characterization of a Rab11 homologue, EoRab11a, in Euplotes octocarinatus.
Rab GTPases are crucial in the regulation of intracellular vesicular trafficking. A novel Rab GTPase gene, EoRab11a (GenBank accession no. EF061065), was isolated and identified from Euplotes octocarinatus cells in this study. It contains an ORF of 696-bp nucleotides, encoding 231 amino acids with a calculated molecular weight of 26.8 kDa. Alignment of EoRab11a with other Rab11 proteins from other eukaryotes demonstrated that these proteins shared 53-61% identity at the amino acid level. The recombinant EoRab11a was expressed in Escherichia coli and purified by immobilized metal chelate affinity chromatography and iron chromatography. The GTPase activity of EoRab11a was 0.0024 min(-1) detected by HPLC at 30 degrees C. Three mutations were generated at amino acids Ser21 and Gly22 positions in the G1 domain of EoRab11a. All three mutants, S21P, S21G and G22R, increased the GTPase activity in vitro. Immunofluorescence microscopy results indicated that EoRab11a was localized on the phagosomal membrane during phagocytosis of E. octocarinatus. These data show that EoRab11a possesses GTP hydrolysis activity and may participate in vesicle transport events during phagocytosis of E. octocarinatus.
Li
Jiangjiao
J
Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Biotechnology, Shanxi University, Taiyuan, Shanxi, China.
Nie
Yu
Y
Dang
Xuhong
X
Liang
Aihua
A
Chai
Baofeng
B
Wang
Wei
W
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D016812:Euplotes / Q000201:enzymology* / Q000235:genetics*; D006160:Guanosine Triphosphate / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D016366:Open Reading Frames; D010588:Phagosomes / Q000737:chemistry; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D020691:rab GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000099193:rab11 GTP-Binding Proteins
D016054:DNA, Protozoan; D006160:Guanosine Triphosphate; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins