Characterization and Cellular Localization of δ-tubulin from Tetrahymena thermophila
XU Jing, WANG Wei* , LIANG Ai-Hua
(Institute of Biotechnology, Key Laboratory of Chemical Biology and Molecular Engineering, Ministry of Education, Shanxi University, Taiyuan030006, China)
Abstract:Tubulin is a highly conserved heterodimeric protein comprised of monomeric globular polypeptides of α- and β-tubulins. Besides γ-tubulin, which is required to initiate the assembly of α-β tubulin heterodimers to form microtubule polymers, 4 new members of tubulin superfamily (δ-,ε-,ζ-, and η-tubulin) were indentified. In this study, the δ-tubulin gene from Tetrahymena thermophia (Tetrahymena delta tubulin 1, TDT1, TTHERM_00335970, http://www. ciliate. org) was isolated. Two different transcripts, 1 326 bp and 1 363 bp, were obtained by RT-PCR. The 1 326 bp transcript encodes 441 amino acids; the 1363 bp transcript is a frameshift mutant containing a 37 bp intron. Quantitative reverse transcriptase PCR (qRTPCR) analysis showed that TDT1 was expressed in both the growing and conjugation stage with higher levels in the latter. HA-tagged TDT1 localizeds at the basal bodies, macronuclei, micronuclei and conjugation junction. The TDT1 knockout strains showed that neo4 cassette could not fully replace the macronuclear TDT1 with increasing paromomysin concentration. The results indicated that TDT1 was a multifunctional protein for normal functions of Tetrahymena cells.