Functional Analysis Of Ran-binding Protein1from Tetrahymena Thermophila

Ran belongs to the family of small G proteins. Ran has intrinsic GTPase activity and regulates a variety of cellular processes including chromosome stability, mitotic spindle microtubule assembly, formation of the nucleus and nucleocytoplasmic transport, by the cycling between active RanGTP and inactive RanGDP.Ran-binding Protein1（Rbp1p）, which facilitates activation of Ran GTPase, is a necessary regulator of the Ran GTPase cycle. Ran-binding Protein1contains a conservative Ran-binding domain （RBD）, which can bind with Ran to promote the hydrolysis of the Ran-GTP complex. In mammalian cells, Ran-binding Protein1was an important regulatory factor that coordinates multiple cellular activities:interphase nucleocytoplasmic transport, centrosome assembly, mitotic spindle microtubule assembly, nuclear envelope reassembly, and even played an important role in apoptosis.Tetrahymena thermophila, the ciliated protozoan, is the good model organisms to research nucleus dynamic changes. T. thermophila contains two differently functioning nuclei:the somatic macronucleus which is responsible for all gene expression, and the germline micronucleus which remains silent during vegetative growth and stores and transmits genetic information during sexual reproduction. RAN1gene （TTHERM00023980） encodes putative Ran GTPase is sustained high expression throughout all stages of T. thermophila life cycle. In our previous research, Ran1might regulate intramacronuclear microtubule assembly and coordinate macronuclear amitosis. Currently, there are no related researches to explore the function of Rbp1p in T. thermophila.In this study, we identified an evolutionarily conservative Ran-binding Protein1gene termed RBP1（TTHERM₀0158040, http://www.ciliate.org） from T. thermophila. Cellular localization and functions of Rbplp were analyzed, the results are as follows: 1. Bioinformatics analysis of RBP1. RBP1（TTHERM₀0158040） was about558bp, and encoded a predicted protein of185amino acids （aa） harboring a single RBD domain. Real-time PCR showed that the RBP1was expressed in growing, starvation and conjugation stage, and the expression level upregulated in conjugation stage. This result was similar to the expression profiling of RBP1in Tetrahymena Functional Genomics Database （http://tfgd.ihb.ac.cn）. Sequence comparison showed that Rbplp contained a conserved Ran-binding domain （RBD）, and have a similar molecular evolution to Rani.2. The immunofluorescence localization of Rbplp. HA-RBP1was constructed and transformed into Wild-type Cu428and B2086cells, respectively. Transformed cells were selected by paromomycin and identified by PCR. Immunofluorescence localization of HA-Rbplp showed that Rbplp was specially localized in the cytoplasm during vegetative growth. During early conjugation stage, Rbp1p localized in the cytoplasm and without nuclear localization; while at the period of "anlagen", Rbp1p localized both in cytoplasm and the old macronucleus, until the old macronucleus completely apoptosis.3. Over-expression of Rbplp led to abnormal nuclear division. pXS-RBP1was constructed and transformed into Wild-type Cu428and B2086cells, respectively. Transformed cells were selected by paromomycin and identified by PCR. In the recombinant cells, the RBP1recombination into MTT1locus, so that RBP1gene could be induced by Cd2+to express Rbplp. The results showed that cell growth rate decreased in the recombinant cells, and over-expression of Rbplp caused abnormal macronuclear amitosis and multi-micronuclear cells. Meanwhile, the abnormal phenotype dependent on the over-expression levels of the Rbplp.4. Knockdown of RBP1led to abnormal macronuclear amitosis. pNeo-RBP1was constructed and transformed into Wild-type Cu428and B2086cells, respectively. Transformed cells were selected by paromomycin and identified by PCR. In the recombinant cells, Neo4gene partial substitute for RBP1gene. Incomplete somatic knockout of RBP1resulted in aberrant intramacronuclear microtubule array formation, missegregation of macronuclear chromosomes and ultimately blocked macronuclei proliferation.Taken together, we identified an evolutionarily conservative Ran-binding Protein1gene from T. thermophila. Over-expression or insufficient expression of Rbplp caused abnormal macronuclear amitosis or formed multi-micronuclear cells. Rbp1p could regulate Ran GTPase pathway, which is involved in assembly of a specialized intramacronuclear microtubule network and coordinates amitotic progression in T. thermophila. Rbplp affects the nuclear division and its normal expression plays an important role in T. thermophila.