Abstract
The majority of the alpha-tubulin in cultured neurons is acetylated (Black and Keyser, 1987). The present studies examine the relationships of the acetylation and deacetylation reactions to tubulin assembly and disassembly in intact neurons. Extraction assays which separate assembled and unassembled tubulin pools reveal that greater than or equal to 99% of the total acetylated, as well as newly acetylated, tubulin is cytoskeletal associated. Treatment of neurons with depolymerizing drugs results in a progressive decrease in the levels of total tubulin in polymer and a corresponding increase in the levels of soluble tubulin. These drugs also cause a progressive decrease in the levels of acetylated alpha-tubulin in polymer that closely parallels in rate and extent that of total alpha-tubulin. However, there is no corresponding increase in soluble acetylated tubulin. Because the total levels of alpha-tubulin remain unchanged during drug treatment, the decrease in levels of acetylated alpha-tubulin during depolymerization must reflect its rapid conversion to nonacetylated alpha-tubulin. These findings suggest alpha-tubulin is acetylated in the polymeric form and that deacetylation is closely coupled to depolymerization. The close coupling between alpha-tubulin deacetylation and depolymerization provided a means of estimating the rate at which subunits cycle off microtubules in intact neurons. Acetate turnover on tubulin in intact neurons was determined both by pulse-chase protocols with 3H-acetate and by measuring the loss of acetylated subunits (using quantitative immunoblotting) under conditions of net microtubule depolymerization induced by colchicine. Both methods yielded similar results. Acetate turnover occurred biphasically; 30–50% of the acetate on tubulin turns over with a t1/2 of 1.5–2 hr, and the remaining half or more turns over with a t1/2 of 5–10 hr. We suggest that these kinetically distinguishable pools of acetylated alpha-tubulin reflect distinct pools of acetylated microtubules that differ in their average rates of subunit turnover.