Characterization of a Rab11 homologue, EoRab11a, in Euplotes octocarinatus |
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Authors: | Jiangjiao Li,Yu Nie,Xuhong Dang,Aihua Liang,Baofeng Chai,& Wei Wang |
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Affiliation: | Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Biotechnology, Shanxi University, Taiyuan, Shanxi, China;and;Research Center for Cardiovascular Regenerative Medicine, The Ministry of Health, Cardiovascular Institute and Fuwai Hospital, Peking Union Medicine College &Chinese Academy of Medical Sciences, Beijing, China |
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Abstract: | 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 . |
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Keywords: | EoRab11a Euplotes octocarinatus GTPase activity phagocytosis |
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