POLYMERIZATION OF ANTARCTIC FISH TUBULINS AT LOW-TEMPERATURES - ROLE OF CARBOXY-TERMINAL DOMAINS

We have proposed previously that the efficient polymerization of tubul ins from Antarctic fishes at low, physiological temperatures (-1.8 to +2 degrees C) may result in part from adaptations (e.g., reductions in acidic residues) located in their carboxy-terminal (C-terminal) tails [Detrich & Overton (1986) J. Biol. Chem. 261, 10922-10930]. To test t his hypothesis, we have examined the polymerization of Antarctic fish neural tubulins modified at their C termini by proteolysis or by neutr alization of carboxyl groups. Addition of subtilisin to low concentrat ions of Notothenia coruceps tubulin induced a biphasic assembly reacti on: stage I corresponded to the C-terinal cleavage of beta chains to p roduce alpha beta(s) dimers, and stage II coincided with the slower, C -terminal cleavage of a chains to yield alpha(s) beta(s). Both stage I and stage II polymers consisted of protofilament sheets and microtubu les with attached sheets. The critical concentration for assembly of t he stage II polymer was at least 10-fold lower than that of untreated tubulin. Neutralization of Glu and Asp carboxyls in Gobionotothen gibb erifrons microtubules by the carbodiimide-catalyzed incorporation of g lycine ethyl ester (GEE) moieties produced a tubulin, modified largely in its C termini, that assembled more readily than did control tubuli n. When 12 GEE groups were incorporated per dimer, the critical concen trations for assembly of modified tubulin at 5-10 degrees C were 2-3-f old smaller than those for the unmodified protein. Comparably modified bovine tubulin (10 GEE/dimer) assembled at 37 degrees C with a critic al concentration 2.6-fold lower than that for the unmodified tubulin. GEE-modified G. gibberifrons tubulin, like modified bovine tubulin, pr oduced microtubules which were indistinguishable from those formed by control tubulin. Our results suggest that the C termini of Antarctic f ish tubulin are not major loci for cold adaptation of microtubule asse mbly.