Purification and reconstitution of HeLa cell microtubules

Microtubules from suspension cultures of HeLa cells have been purified by carrying them through four complete cycles of polymerization at 37 degrees C and depolymerization at 4 degrees C. These microtubules show, in addition to the major alpha- and beta-tubulin components, major proteins with molecular weights of 201 000-206 000 (comprising 4.5% of the total protein), proteins with molecular weights of 97 000, 100 000, 104 000, and 114 000 (together comprising approximately 2% of the total protein), and minor components with molecular weights of 68 000 and 151 000. HeLa microtubules have also been reconstituted from purified HeLa tubulin and proteins from HeLa microtubules separated from tubulin by DEAE-cellulose column chromatography. Experiments on the fractionation and reconstitution of both two- and four-cycle microtubules suggest that the 201 000-206 000-dalton proteins are incorporated into microtubules and promote tubulin polymerization. Microtubules formed by fractionationand reconstitution of two-cycle microtubules also contain several other proteins with molecular weights of 132 000, 146 000, 151 000, 160 000, and 284 000, although these are not present in microtubules carried through four assembly-disassembly cycles. Evidence is also presented which shows that a 68 000-dalton protein which is a prominent component of HeLa microtubules after two polymerization-depolymerization cycles does not stoichiometrically copurify with tubulin through repeated assembly--disassembly cycles and does not stimulate tubulin polymerization. On the other hand, the sedimentation of this 68 000-dalton protein is apparently influenced by the presence of polymerized microtubules, suggesting that this protein may be a component of a system whjich interacts weakly with microtubules. Finally, evidence is presented suggesting that two-cycle microtubules contain a proteolytic activity that can digest the 201 000-206 000-dalton proteins.