The highly divergent α- and β-tubulins from Dictyostelium discoideum are encoded by single genes

Abstract

As a step in the characterization of the microtubule system of Dictyostelium discoideum, we have isolated and sequenced full-length cDNA clones that encode the Dictyostelium alpha- and beta-tubulins, as well as the Dictyostelium alpha-tubulin gene. Southern blot analysis suggests that Dictyostelium is unusual in that its genome contains single alpha- and beta-tubulin genes, rather than the multi-gene family common in most eukaryotic organisms. The complete alpha-tubulin cDNA contains 1558 nucleotides, with an open reading frame, that encode a protein of 457 amino acids. The complete beta-tubulin cDNA contains 1572 nucleotides and encodes a protein of 456 amino acids. Analysis of the deduced protein sequences indicates that while there is a significant degree of sequence similarity between the Dictyostelium tubulins and other known tubulins, the Dictyostelium alpha-tubulin displays the greatest sequence divergence yet described. Single alpha- and beta-tubulin transcripts are detected by northern blot analysis during all stages of Dictyostelium development. The highest levels of message accumulate late in germinating spores and vegetative amoebae. Despite changes in alpha- and beta-tubulin mRNA levels, protein levels remain constant throughout development. We have expressed the carboxy-terminal two-thirds of the alpha- and beta-tubulins as trpE fusions in Escherichia coli and used this protein to produce polyclonal antisera specific for the Dictyostelium alpha- and beta-tubulins. These antisera recognize one alpha- and two beta-tubulin spots on western blots of 2-D gels and, by indirect immunofluorescence, both recognize the interphase and mitotic microtubule arrays in vegetative amoebae.

Phosphofructokinase from Dictyostelium discoideum Is a Potent Inhibitor of Tubulin Polymerization

“…D. discoideum α - and β -tubulins are among the most divergent members of the highly conserved family of tubulins (). We did not test the action of DdPFK on D. discoideum tubulin, since this protein has not been successfully purified from the slime mold because of its low content and the occurrence of high levels of proteases and tubulin inhibitors, as already pointed out ( , ). However, the interaction of DdPFK with bovine brain tubulin devoid of the hypervariable acidic C-termini suggests that it can bind D. discoideum tubulin as well.…”

“…However, a transient formation of a microtubule array during interphase has also been described in the slime mold cytoplasm (57), therefore requiring the action of tubulin regulators as well. D. discoideum Rand β-tubulins are among the most divergent members of the highly conserved family of tubulins (58). We did not test the action of DdPFK on D. discoideum tubulin, since this protein has not been successfully purified from the slime mold because of its low…”

“…content and the occurrence of high levels of proteases and tubulin inhibitors, as already pointed out (57,58). However, the interaction of DdPFK with bovine brain tubulin devoid of the hypervariable acidic C-termini suggests that it can bind D. discoideum tubulin as well.…”

Toxin-Producing Endosymbionts Shield Pathogenic Fungus against Micropredators

“…S1E ), which is an important feature in rhizoxin-sensitive fungi ( 32 ). Depolarization of microtubule in amoeba has severe consequences, as β-tubulins have been shown to be essential parts of the microtubule network in D. discoideum , important for cell polarity, migration, and the movement of intracellular particles ( 50 ). Intriguingly, P. aurantium requires substantial condensation of actin filaments when grabbing and invading the rather large fungal hyphae ( 15 ).…”