Localization And Functional Analysis Of RanGTPpase Activating Protein (RanGAP) From Tetrahymena Thermophila

RanGTPase-activating protein （RanGAP） is an important factor which regulates the activity of Ran in cytoplasm. RanGAP has a leucine-rich repeats domain （LRRs Domain）. It interacts with Ran to promote the hydrolysis of Ran_GTP. Mammalian RanGAP takes part in nucleocytoplasmic transport, mitotic spindle formation, and nuclear envelope assembly during mitosis. RanGAP is required for the post-meiotic mitosis in female gametophyte development in Arabidopsis thaliana. RanGAP participates in heterochromatin assembly in Saccharomyces cerevisiae.Tetrahymena thermophila is an eukaryotic unicellular protozoan. The single cell contains a germline micronucleus and a somatic macronucleus. Tetrahymena is a good system to explore the molecular mechanism of the nuclear dynamic change. Ran, Ran binding protein, and RCC1 are involved in the macronuclear amitosis in Tetrahymena..In this study, we first identified and characterized a novel RanGAP （TTHERM₀0766430） from T.thermophila. The results are as follows:1. Bioinformatic analysis of RanGAP. RanGAP （TTHERM₀0766430） was 1423bp, contains two introns. Open reading frame was 1074bp and encoded 357 amino acids. RanGAP contains a conservative leucine-rich repeats domain （LRRs）. RanGAP was expressed in vegetative growth, starvation and conjugation stage. Specifically, RanGAP has a higher express level at 4-6 h during conjugation stage. Sequence alignment of RanGAPs from different species showed that evolution of conservative RanGAP is consisted with species evolution.2. Subcellular localization of HA-RanGAP. The vector pNeo4-HA-RanGAP was constructed and transformed into wild-type Tetrahymena. HA-RanGAP cells were selected by paromomycin and identified by PCR. Immunofluorescence showed that HA-RanGAP was localized in the cytoplasm during vegetative growth, starvation, and conjugation stage. Furthermore, HA-RanGAP was also occured in the apoptotic parental macronuclei and staggered with DNA during late conjugation stage. Western blotting analysis showed that two different sizes bands.40kD is HA-RanGAP,48kD could be HA-RanGAP-X which was SUMOlated modification. With parental macronuclear apoptosis, the relative expression level of HA-RanGAP-X increased. These result indicating that the sumolated RanGAP could be involved in the parental macronuclear apoptosis in Tetrahymena.3. RanGAP invovled in macronuclear amitosis and cytokinesis. The overexpression vector pXS-RanGAP and knockout vector pNeo4-KO-RanGAP was constructed and transformed into Wild-type Tetrahymena cells, respectively. RanGAP overexpression cells and RanGAP knockout cells were obtained by paromomycine resistance screening. Overexpression or knockdown of RanGAP inhibited cellular proliferation, led to abnormal macronuclear amitosis and abnormal cytokinesis, produced amacronuclear cell.4. RanGAP affect RAN1 signal pathway. Overexpression of RanGAP led to down regulation of RAN1, RBP1, and RCC1. In contrast, knockdown of RanGAP led to upregulation of RAN1,RBP1, and RCC1 in Tetrahymenay. The results indicated that RanGAP is member of Tetrahymena Ran signal pathway and the expression of the factors is likely to be transcriptionally regulated by each other.Taken together, we identified RanGAP from Tetrahymena. HA-RanGAP was localized in the cytoplasm during vegetative growth, starvation and conjugation stage. HA-RanGAP also occurs in the apoptotic parental macronuclei during late conjugation stage. Overexpression or knockdown of RanGAP led to abnormal macronuclear amitosis. Ran GAP is key member of Tetrahymena Ran signal pathway. RanGAP regulated the macronuclear amitosis by Ran pathway and could be involved in the parental macronuclar apoptosis in Tetrahymena.