Biochemical dissection of the role of the one-kilodalton carboxyl-terminal moiety of tubulin in its assembly into microtubules

Abstract
The 4-KDa C-terminal domain of both tubulin subunits plays a major role in the regulation of microtubule assembly [Serrano et al. (1984) Biochemistry 23, 4675]. Controlled proteolysis of tubulin with subtilisin produces the selective cleavage of this 4-kDa moiety from .alpha.- and .beta.-tubulin with a concomitant enhancement of the assembly. Here we show that gradual removal of the last six to eight amino acid residues of the C-terminal region of .alpha. and .beta. subunits by an exopeptidase, carboxypeptidase Y, produces a modified protein (C-tubulin) without relieving the modulatory effect of the C-terminal domain and the usual need of MAPs for microtubule assembly. Actually, treatment with this proteolytic enzyme did not change tubulin assembly as promoted by either MAP-2, taxol, MgCl2, dimethyl sulfoxide, or glycerol. The critical concentration for the assembly of C-tubulin remained the same as that for the unmodified tubulin control. Microtubule-associated proteins MAP-2 and .tau. incorporated into C-tubulin polymers. Clearly, pure C-tubulin did not assemble in the absence of MAPs or without addition of assembly-promoting compounds. However, proteolysis with the exopeptidase induced changes in tubulin conformation as assessed by biophysical methods and double-limited proteolysis. The cleavage with subtilisin after carboxypeptidase digestion did not result in enhancement of the assembly to the levels observed after the treatment of native tubulin with subtilisin. Interestingly, Ca2+ ions affected neither C-tubulin assembly nor depolymerized microtubules assembled from C-tubulin. These findings indicate that the very end C-terminal moiety which has been shown to be the region that defines tubulin isotypes, is not directly involved in the selective interactions of MAPs. Instead, this region appears to be essential for the conformation of native tubulin and also for its sensitivity to Ca2+ inhibition.