IDENTIFICATION OF THE MAJOR 68000-DALTON PROTEIN OF MICROTUBULE PREPARATIONS AS A 10-NM FILAMENT PROTEIN AND ITS EFFECTS ON MICROTUBULE ASSEMBLY INVITRO

The major 68 000-dalton protein present in cycled microtubule preparations from bovine brain can be isolated in a rapidly sedimenting fraction consisting of filaments 10 nm in diameter. This 68 000-dalton protein remains in the filament fraction after gel filtration, phosphocellulose chromatography, or salt extraction of microtubule protein. Microtubule protein devoid of 10-nm filaments contains ring structures under depolymerizing conditions, and it polymerizes into microtubules with a characteristically low critical concentration, although all of the 68 000-dalton protein has been removed from it. When cycled microtubule protein is subjected to chromatography on phosphocellulose, the tubulin fraction (PC-tubulin) assembles into microtubules only at concentrations greater than 2 mg/mL. The other fraction, eluted from phosphocellulose at high ionic strength, contains the major 68 000-dalton protein and can be further resolved into wo components by centrifugation. The supernatant, which consists mainly of high molecular weight microtubule-asso-ciated proteins, stimulates low concentrations of PC-tubulin to assemble. The pellet contains all of the 68 000-dalton protein, consists of 10-nm filaments, and does not stimulate assembly of PC-tubulin. Boiling of purified filaments, however, releases several proteins, including the 68 000-dalton protein, and these released proteins stimulate the assembly of PC-tubulin. The morphology and protein composition of the filaments isolated from microtubule preparations by these techniques are very similar to those of mammalian neurofilaments. These results suggest that the major 68 000-dalton protein in cycled microtubule preparations, which may correspond to tubulin assembly protein [Lockwood, A. H. (1978) Cell 13, 613-627], is a constituent of neurofilaments. © 1979, American Chemical Society. All rights reserved.