Multiple sites for subtilisin cleavage of tubulin: effects of divalent cations

Limited digestion of pig brain GDP-tubulin by subtilisin was carried out in the presence of Mg2+, Mn2+, Ca2+, Zn2+, or Be2+. Isoelectric focusing, followed by SDS-PAGE, revealed characteristic divalent cation-dependent changes in the alpha- and beta-tubulin cleavage patterns. Previous studies revealed that the beta-cleavage pattern is different for heterodimers and microtubules [Lobert and Correia, 1992: Arch. Biochem. Biophys. 296: 152-160]. Divalent cation effects on subtilisin digestion of tubulin indicate different classes of divalent cation binding sites. Western blot analysis locates the proteolytic zone at residue 430 or higher in both subunits for all conditions. Turbidity and electron microscopy reveal that GDP-tubulin cleaved by subtilisin in the presence of Mg2+, Ca2+, or Mn2+ forms sheets of rings. Mn2+ induces ring formation in uncleaved GDP-tubulin. Isotype-depleted tubulin was generated by the removal of class III beta-tubulin using immunoaffinity chromatography. Subtilisin digestion of the depleted fraction and the purified class III beta-tubulin demonstrates that cleavage occurs at three to four distinct sites. Thus, subtilisin-digested tubulin is more heterogeneous than was previously reported and the cleavage sites depend on solution conditions, divalent cations, and the state of assembly. This has important implications for experiments that utilize subtilisin-digested tubulin for studying microtubule-associated protein binding.