Posttranslational modifications and assembly characteristics of goldfish tubulin.

Abstract

Cell-free extracts from goldfish brain, ovarian follicles, testes and the cell line, ATCC CCL-71, were analyzed for posttranslationally modified tubulins. All samples, with the exception of that from brain where the reverse was true, contained more tyrosinated than detyrosinated alpha-tubulin. Additionally, extracts from brain and testes exhibited acetylated alpha-tubulin whereas this isoform was not visualized on blots of cell-free preparations from follicles and CCL-71 cells. Assembly of brain and ovary tubulin was induced with taxol. Brain tubulin, partially purified through three cycles of assembly/disassembly was associated with a variety of putative microtubule-associated proteins (MAPs), most of which had a high molecular mass. There were very few cold stable microtubules in brain preparations whereas, for ovary, purification of tubulin was hampered by significant losses of cold stable polymer. Comparison of brain and ovary showed there was no correlation between the extent of alpha-tubulin detyrosination or acetylation and cold stability of microtubules. Moreover, cycled tubulin from ovary contained acetylated tubulin even though this was not observed on blots of cell-free extracts from ovary or from follicles. Cultured goldfish cells contained extensive arrays of microtubules, many of which originated from discrete organizing centers. The results reveal the widespread distribution of posttranslationally modified tubulins in goldfish tissues, the differing assembly/disassembly characteristics of tubulin from brain versus ovary, and the presence of putative neural MAPs, mostly with a high molecular mass.