Functional analysis of microtubule-binding domain of bovine MAP4

Bovine microtubule-associated protein 4 (MAP4) consists of an amino-termina l projection domain and a carboxyl-terminal microtubule-binding domain. The carboxyl-terminal domain of MAP4 is further divided into three subdomains: a region rich in proline and basic residues (Pro-rich region), a region co ntaining four repeats of an assembly-promoting (AP) sequence, which consist s of 22 amino acid residues (AP sequence region), and a hydrophobic tail re gion (Tail region). The subdomain structure of MAP4 microtubule binding dom ain is similar to those of other MAPs (MAP2 and tau). In order to study the function of each subdomain per se of bovine MAP4 micr otubule-binding domain, we purified a series of truncated fragments of MAP4 , expressed in Escherichia coli. Binding affinity of the PA(4)T fragment (c ontaining the Pro-rich region, the AP sequence region and the Tail region) is only four times higher than that of the A(4)T fragment (containing the A P sequence region and the Tail region), while the microtubule nucleating ac tivity of the PA4T fragment is far greater. We propose that the Pro-rich re gion promotes the nucleation of microtubule assembly. The A(4) fragment (corresponding to the AP sequence region) stimulated the assembly of tubulin into cold-stable amorphous aggregates. The AP sequence region of MAP4 failed to promote microtubule assembly. On the other hand, t he fragment has an activity to stimulate microtubule elongation. The function of the MAP4 Tail region is not clear at present. The AIT fragm ent (containing the AP sequence region and the Tail region) promote both mi crotubule nucleation and elongation step, but the A(4) fragment only promot es microtubule elongation, suggesting that the Tail region is indispensable for the nucleation step. However, the fragment containing only the Tail re gion could not bind to microtubule. Although MAP4 was considered to be long , thin and flexible molecule, never the Tail region may contribute to be th e proper folding of MAP4, and/or may interact with other molecules. We concluded that both the Pro-rich region and the AP sequence region take part in the promotion of tubulin polymerization, and that the former is imp ortant for the lateral protofilament-protofilament interaction, and the lat ter is important for the longitudinal affinity between each tubulin dimer i n a protofilament.