Participation of 200K or 150K subunit of neurofilament in construction of the filament core with 70K subunit and promotion of tubulin polymerization by incorporated 200K subunit.

We have already reported that neurofilaments are capable of stimulating microtubule assembly and causing gelation. After separation of each of the triplet proteins of neurofilaments it was demonstrated that only the 200K subunit shows the activity to promote tubulin polymerization (Minami, Y. & Sakai, H. (1983) J. Biochem. 94, 2023-2033). The separation of each subunit protein led us to attempt the reconstitution of filaments from the 200K and 70K subunits or from the 150K and 70K subunits. It was found that both the 200K and 150K subunits independently contribute to the formation of intermediate-sized filaments, provided that each subunit was combined with the 70K subunit before removing urea by dialysis for reconstitution. On the other hand, the 200K subunit alone formed a very short thread-like structure after removal of urea, and the 150K subunit formed a filamentous structure, both incapable of being incorporated into filaments made of the 70K subunit alone. These observations suggest that the 200K and 150K subunits are not peripherally attached to a filament core made of 70K protein, but they take part in the formation of the core. Moreover, both proteins can co-polymerize with the 70K protein at a weight ratio of about 1 : 1 at least, which is in excess of that of the intact neurofilament. We investigated whether or not the 200K subunit incorporated with the 70K subunit into filaments could also stimulate tubulin polymerization. Low-shear viscometry measurements suggested that the 200K subunit retains the activity to initiate tubulin polymerization. This was confirmed by measuring viscosity changes with an Ostwald-type viscometer. In contrast, filaments reconstituted from the 70K and 150K proteins were incapable of increasing low-shear viscosity when mixed with tubulin. These observations suggest that the domain of the 200K protein embedded in the core of intermediate-sized filament is separate from the site responsible for promotion of tubulin polymerization.