SEQUENCE-ANALYSIS AND IMMUNOFLUORESCENCE STUDY OF ALPHA-TUBULINS AND BETA-TUBULINS IN RETICULOMYXA-FILOSA - IMPLICATIONS OF THE HIGH-DEGREEOF BETA-2-TUBULIN DIVERGENCE

We have cloned and sequenced 2 alpha- and 2 beta-tubulin isoforms from the giant freshwater amoeba Reticulomyxa filosa. The microtubules of this organism exhibit some unusual properties, including the highest r ates of assembly and disassembly known and the inability to be stabili zed by taxol. The cloned alpha-tubulins show a high degree of identity when compared to an alpha-tubulin consensus sequence. The beta-tubuli ns, however, are more divergent, the beta 2-tubulin being the most unu sual beta-tubulin found so far. The deduced amino acid sequence of bet a 2 shows 55% identity to a beta-tubulin consensus sequence. It also f eatures 51 unique exchanges which cluster in the C-terminal half of th e molecule. Several unique exchanges and two insertions occur3 in regi ons adjacent to, or directly implicated in, conserved beta-tubulin fun ctions. A phylogenetic analysis places the beta-tubulins of R. filosa in the vicinity of beta-tubulins from fungi and slime molds. Monoclona l and polyclonal antibodies raised against R. filosa tubulins show tha t the electrophoretic mobility of alpha- and beta-tubulins is reversed with respect to tubulins from most other sources. Immunofluorescence experiments reveal a ubiquitous distribution of both beta-tubulins in the amoebal network. Our observations suggest possible links between t he aberrant primary structure of the beta 2-tubulin and the unusual pr operties of R. filosa microtubules. (C) 1997 Wiley-Liss, Inc.