Identification of beta(III)- and beta(IV)-tubulin isotypes in cold-adaptedmicrotubules from Atlantic cod (Gadus morhua): Antibody mapping and cDNA sequencing

Isolated microtubule proteins from the Atlantic cod (Gadus morhua) assemble at temperatures between 8 and 30 degrees C. The cold-adaptation is an intr insic property of the tubulin molecules, but the reason for it is unknown. To increase our knowledge of tubulin diversity and its role in cold-adaptat ion we have further characterized cod tubulins using alpha- and beta-tubuli n site-directed antibodies and antibodies towards posttranslationally modif ied tubulin. In addition, one cod brain beta-tubulin isotype has been seque nced. In mammals there are five beta-tubulins (beta(I), beta(II), beta(III) , beta(IVa) and beta(IVb)) expressed in brain. A cod beta(III)-tubulin was identified by its electrophoretic mobility after reduction and carboxymethy lation. The beta(III)-like tubulin accounted for more than 30% of total bra in beta-tubulins, the highest yield yet observed in any animal. This tubuli n corresponds most probably with an additional band, designated beta(x), wh ich was found between alpha- and beta-tubulins on SDS-polyacrylamide gels. It was found to be phosphorylated and neurospecific, and constituted about 30% of total cod beta-tubulin isoforms. The sequenced cod tubulin was ident ified as a beta(IV)-tubulin, and a beta(IV)-isotype was stained by a C-term inal specific antibody. The amount of staining indicates that this isotype, as in mammals, only accounts for a minor part of the total brain beta-tubu lin. Based on the estimated amounts of beta(III)- and beta(IV)-tubulins in cod brain, our results indicate that cod has at least one additional beta-t ubulin isotype and that beta-tubulin diversity evolved early during fish ev olution. The sequenced cod beta(IV)-tubulin had four unique amino acid subs titutions when compared to beta-tubulin sequences from other animals, while one substitution was in common with Antarctic rockcod beta-tubulin. Residu es 221, Thr to Ser, and 283, Ala to Ser, correspond in the bovine tubulin d imer structure to loops that most probably interact with other tubulin mole cules within the microtubule, and might contribute to cold-adaptation of mi crotubules. Cell Motil. Cytoskeleton 42:315-330, 1999. (C) 1999Wiley-Liss,I nc.