Identification And Characterization Of Rab-GTPase Involved In Vesicle Traffic In Euplotes

Rab proteins are key regulators of vesicular traffic in eukaryotic cells. It belongs to the largest family of the Ras superfamily of small GTPase that plays an important role in intracellular vesicular traffic. Ciliate, a unicellular protozoa and low eukaryotes in evolution, has perfect vesicular trafficking system among organelles. Identification and characterization of the Rab gene from ciliate would facilitate to understand the mechanism of vesicular transportation.To further study the diversity and function of Rab protein in evolution, unicellular protozoa ciliates, Euplotes octocarinatus, were used in this paper. Rab genes were screened by PCR method from macronuclear DNA and micronuclear DNA of Euplotes octocarinatus, respectively. Sixteen Rab genes named from Eorablb to Eorablu were obtained from macronuclear DNA. They share 87.6-99.5% identities. Highly conserved GTP-binding domains were found. The macronuclear DNA molecule carrying the Rab gene is 767 bp long and shows characteristics typical of macronuclear chromosomes of hypotrichous ciliates. Three of the five cysteines are encoded by the opal codon TGA. The deduced protein is a 207-amino acid (aa) with a molecular mass of 23 kDa.A micronuclear version of the Rab gene was amplified by polymerase chain reaction (PCR). Analysis of the sequences indicated that the micronuclear version of the Rab gene contains two internal eliminated sequences, IES1 and IES2. IES1 is flanked by a pair of hepta-nucleotide 5'-AAATTTT-3' direct repeats, and IES2 is flanked by 5'-TA-3' direct repeats. The DNA in a micronucleus undergoes remarkable rearrangements when it develops into a macronucleus after cell mating in the hypotrichous ciliate.In order to understand the function of the Rab protein in ciliates, the...