Characterization of a Rab11 homologue, EoRab11a, in Euplotes octocarinatus

• Published in: FEMS microbiology letters Vol. 292; no. 2; pp. 222 - 230
• Main Authors: Li, Jiangjiao, Nie, Yu, Dang, Xuhong, Liang, Aihua, Chai, Baofeng, Wang, Wei
• Format: Journal Article
• Language: English
• Published: Oxford, UK Oxford, UK : Blackwell Publishing Ltd 01.03.2009; Blackwell Publishing Ltd; Oxford University Press
• Subjects: Affinity chromatography; Amino Acid Sequence; Amino Acid Substitution - genetics; Amino acids; Animals; Base Sequence; Chelates; Chromatography; Chromatography, Affinity; Chromatography, High Pressure Liquid; DNA, Protozoan - chemistry; DNA, Protozoan - genetics; E coli; EoRab11a; Escherichia coli; Escherichia coli - genetics; Eukaryotes; Euplotes; Euplotes - enzymology; Euplotes - genetics; Euplotes octocarinatus; Gene expression; GTPase activity; Guanosine triphosphatases; Guanosine triphosphate; Guanosine Triphosphate - metabolism; High performance liquid chromatography; Homology; Immunofluorescence; Liquid chromatography; Microbiology; Molecular Sequence Data; Molecular Weight; Mutagenesis, Site-Directed; Mutation; Mutation, Missense; Nucleotides; Open Reading Frames; Phagocytosis; Phagosomes - chemistry; Proteins; Protozoan Proteins - chemistry; Protozoan Proteins - genetics; Protozoan Proteins - metabolism; rab GTP-Binding Proteins - chemistry; rab GTP-Binding Proteins - genetics; rab GTP-Binding Proteins - metabolism; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Sequence Alignment; Sequence Analysis, DNA; Sequence Homology, Amino Acid; phagocytosis; GTPase activity; EoRab11a
• ISSN: 0378-1097; 1574-6968
• DOI: 10.1111/j.1574-6968.2009.01485.x

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 °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.