Role of the carboxy-termini of tubulin on its chaperone-like activity

Mutational analysis and the enzymatic digestion of many chaperones indicate the importance of both hydrophobic and hydrophilic residues for their uniq ue property. Thus, the chaperone activity of alpha -crystallin is lost due to the substitution of hydrophobic residues or upon enzymatic digestion of the negatively charged residues. Tubulin, an eukaryotic cytoskeletal protei n, exhibits chaperone-like activity as demonstrated by prevention of DTT-in duced aggregation of insulin, thermal aggregation of alcohol dehydrogenase, beta gamma -crystallin, and other proteins. We have shown that the tubulin lost its chaperone-like activity upon digestion of its negatively charged C-termini. In this article, the role of the C-terminus of individual subuni ts has been investigated. We observe that the digestion of C-terminus of be ta -subunit with subtilisin causes loss of chaperone-like activity of tubul in. The contribution of C-terminus of alpha -subunit is difficult to establ ish directly as subtilisin cleaves C-terminus of beta -subunit first. This has been ascertained indirectly using a 14-residue peptide P2 having the se quence corresponding to a conserved region of MHC class I molecules and tha t binds tightly to the C-terminus of a-subunit. We have shown that the bind ing of P2 peptide to alpha beta -tubulin causes complete loss of its chaper one-like activity. NAM and gel-electrophoresis studies indicate that the P2 peptide has a significant higher binding affinity for the C-terminus of al pha -subunit compared to that of beta -subunit. Thus, we conclude that both the C-termini are necessary for the chaperone-like activity of tubulin. Im plications for the chaperone functions in vivo have been discussed. (C) 200 1 Wiley-Liss, Inc.