Characterization Of EoRab11a In Euplotes Octocarinatus

The Rab family is the biggest part of the Ras superfamily of small GTPases.Rab proteins have emerged as essential regulators for vesicle trafficking processes,including vesicle budding,motility and fusion, regulating endocytosis,exocytosis and membrane recycling.Different Rab proteins localize in distinct subcellular organelles where they regulate distinct vesicular transport processes.The Rab11 subfamily,an important member of the Rab family,has been shown to be involved in various vesicle trafficking processes,such as receptor recycling,cytokinesis,protein secretion,phagocytosis and signal transduction,which are essential for maintaining normal cellular functions.The Rab11 homologues displayed different localizations and functions in different organisms.It is interesting to explore new functions of Rab11 in evolutionarily different species.Euplotes octocarinatus,a free-living unicellular ciliated protozoan,have elaborate ultrastructure and complicated vesicular membrane trafficking systems,which is a good model organism for functional study of Rabs.In this study,EoRab11a,a Rab11 homologue in E.octocarinatus,was systematically investigated.The results obtained are as follows.1.Cloning and sequence analysis of EoRab11a gene from E. octocarinatus.A novel Rab GTPase gene,EoRab11a(GenBank accession no. EF061065),was obtained from the macronucleus DNA and mRNA of E. octocarinatus by the degenerate PCR.There is no intron in the macronucleus DNA of EoRab11a.The macronucleus DNA carrying EoRab11a is 899 bp, including non-coding regions and telomeric sequences at both ends. EoRab11a gene contains an ORF of 696 bp,encoding 231 amino acids with a calculated molecular weight of 26 kDa.The predicted amino acid sequence has many conserved motifs:GTP-binding sequences,Rab family-specific regions,Rab subfamily specific regions and carboxyl-terminal Cys-Cys motif.The result suggested EoRab11a is a typical Rab family member. 2.Expression and purification of EoRab11a.The EoRab11a ORF was cloned into the expression vector pRSET-C,and the recombinant plasmid pRSET-C-EoRab11a was transformed into E.coli BL21(DE3).The fusion protein His₆-EoRab11a was expressed in E.coli BL21(DE3)/pRSET-C-EoRab11a.His₆-EoRab11a was purified by Ni-NTA sepharose affinity chromatography and Resource-Q weak anion exchange chromatography,and the electrophoretic pure protein was obtained finally. About 20 mg/L protein was obtained.Polyclonal antibody against EoRab11a was prepared by immunization of mice with purified EoRab11a protein. Antibody titer was 1:86000 by ELISA assay,and antibody specificity was validated by Western blotting.3.EoRab11a possesses GTPase activity.The GTP hydrolysis activity of purified EoRab11a protein was examined using HPLC.The intrinsic GTP hydrolysis rate of EoRab11a protein was measured to be 0.0024 min^-1 at 30℃.To investigate the role of the P-loop in GTP hydrolysis of EoRab11a,we have generated three mutants with amino acid substitutions,S21P,S21G and G22R.The GTP hydrolysis rates of S21P, S21G and G22R mutants were 0.0025,0.0043 and 0.0059 min^-1,increasing by 1.04-,1.8- and 2.5-fold,respectively.These data suggested that the P-loop is important to the GTPase activity of EoRab11a,and the changes of amino acid residues in this local structure affected the GTP hydrolysis reaction by an unknown mechanism.4.EoRab11a participates in phagocytosis.The localization of EoRab11a in E.octocarinatus was assessed by immunofluorescence using the antibody against EoRab11a.Dynamic changes in the expression level and localization pattern of EoRab11a were observed in E.octocarinatus.Before ingestion of food,the expression level of EoRab11a was relatively low and localized to a few distinct spots or cycles close to the macronucleus;in the early phagocytosis stage,the expression level of EoRab11a was upregulated and EoRab11a positive vesicles began to encompass the food particles;in the middle phagocytosis,EoRab11a loci were located more closely around some food particles,likely the phagosomal membrane;in the late phagocytosis stage,EoRab11a was positioned adjacent to the macronucleus again when the food particles were finally digested.The results showed that EoRab11a participates in vesicle transport events during phagocytosis of E.octocarinatus.It is reasonable to predict that EoRab11a may play a role in providing sufficient membrane materials for the nascent food vacuole or in delivering digestive enzymes to the food vacuoles to facilitate digestion.5.EoRab11a is involved in cytokinesis.The EoRab11a ORF was cloned into the mammalian expression vector pEGFP-C2.The recombinant plasmid pRSET-C-EoRab11a was transformed into human embryonic kidney cell line 293(HEK293).The fusion protein EGFP-EoRab11a was expressed in HEK293/pRSET-C-EoRab11a.The localization of EoRab11a in HEK293 was observed by fluorescence microscope.In interphase HEK293 cells,EoRab11a was positioned adjacent to the nuclear,which is similar to its distribution in E.octocarinatus.In dividing HEK293 cells,EoRab11a was enriched within the furrow of cells in telophase and present within the midbody.These findings demonstrated that EoRab11a involved in membrane trafficking events of cytokinesis.It is possible that Rab11 is required for delivery of new membrane or required materials to the furrow and midbody and for abscission.In conclusion,the Rab11 protein in E.octocarinatus has been systematically examined in this study.The autofluorescence of C.elongatum, the food of Euplotes,was firstly used in our research to make the colocalization analysis with Rab protein in E.octocarinatus.EoRab11a was found to regulate the phagocytosis in E.octocarinatus.Furthermore,the location of Rab11 in E.octocarinatus was compared with its localization in mammalian cells for the first time.The results suggested that EoRab11a protein maintains similar localization pattern across large evolutionary distances,and this protein is able to function in cytokinesis in HEK293 cells. These findings provide new evidence for understanding the functional conservation of Rab11 family proteins.