Sequence analysis and immunofluorescence study of alpha- and beta-tubulins in Reticulomyxa filosa: implications of the high degree of beta2-tubulin divergence.

Abstract

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 rates of assembly and disassembly known and the inability to be stabilized by taxol. The cloned alpha-tubulins show a high degree of identity when compared to an alpha-tubulin consensus sequence. The beta-tubulins, however, are more divergent, the beta2-tubulin being the most unusual beta-tubulin found so far. The deduced amino acid sequence of beta2 shows 55% identity to a beta-tubulin consensus sequence. It also features 51 unique exchanges which cluster in the C-terminal half of the molecule. Several unique exchanges and two insertions occur in regions adjacent to, or directly implicated in, conserved beta-tubulin functions. A phylogenetic analysis places the beta-tubulins of R. filosa in the vicinity of beta-tubulins from fungi and slime molds. Monoclonal and polyclonal antibodies raised against R. filosa tubulins show that 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 the aberrant primary structure of the beta2-tubulin and the unusual properties of R. filosa microtubules.