Proteolysis of tubulin and the substructure of the tubulin dimer.

The alpha and beta subunits of tubulin each have a single highly reactive site for a variety of proteases that divides each subunit into two unequal regions. The position of cleavage is not the same for alpha and beta, since alpha is consistently cleaved into about 38- and 14-kDa pieces, while beta is cleaved into about 34- and 21-kDa pieces. The larger fragment is amino-terminal in both subunits as shown: by size reduction of the smaller fragment by subtilisin (which cleaves at the extreme carboxyl-terminal end), but no change in size of the larger fragment; by the charge/mass ratios of the proteolytic fragments; and by sequence analysis which locates trypsin cleavage after residue 339 (alpha) and chymotrypsin cleavage after residue 281 (beta). Since this cleavage pattern of the alpha and beta subunits is found for very different proteases, we suggest that it is determined by structural features of the tubulin molecule. The two pieces of each subunit remain associated following cleavage. While both cleavage sites are exposed in the free dimer, assembly of dimers into microtubules or sheets protects the internal site against cleavage. By contrast, the carboxyl-terminal subtilisin-sensitive sites remain exposed. Based on these results we propose a model for the substructure of the tubulin dimer that accommodates internal cleavage in the dimer but not the polymer, access to the COOH termini in both forms, and the orientation of the dimer in the polymer.