Evidence for tubulin-binding sites on cellular membranes: plasma membranes, mitochondrial membranes, and secretory granule membranes.

Open Access
Publisher Website
Google Scholar
Abstract
The interaction was described of pure [rat] brain tubulin with purified membranes specialized in different cell functions, i.e., plasma membranes and mitochondrial membranes from liver and secretory granule membranes from adrenal medulla. The tubulin-binding activity of cellular membranes using a radiolabeled ligand-receptor assay and an antibody retention assay was studied. The tubulin-membrane interaction was time- and temperature-dependent, reversible, specific and saturable. The binding of tubulin to membranes appears to be specific since acidic proteins such as serum albumin or actin did not interfere in the binding process. The apparent overall affinity constant of the tubulin-membrane interaction ranged between 1.5 and 3.0 .times. 107 M-1. Similar values were obtained for the 3 types of membranes. Tubulin bound to membranes was not entrapped into vesicles since it reached quantitatively with antitubulin antibodies. At saturation of the tubulin-binding sites, the amount of reversibly bound tubulin represents 5-10% by weight of membrane protein (0.4-0.9 nmol tubulin/mg membrane protein). The high tubulin-binding capacity of membranes seems to be inconsistent with a 1:1 stoichiometry between tubulin and a membrane component but could be relevant to a kind of tubulin assembly. Tubulin-membrane interaction had some properties in common with microtubule formation. The association of tubulin to membranes increased with the temperature, whereas the dissociation of tubulin-membrane complexes increased by decreasing temperature, the binding of tubulin to membranes was prevented by phosphate buffer. The tubulin-membrane interaction differed from tubulin polymerization in several aspects; it occurred at concentrations far below the critical concentration for polymerization; it was not inhibited at low ionic strength and it was colchicine-insensitive. Plasma membranes, mitochondrial membranes, and secretory granule membranes contained tubulin as an integral component. This was demonstrated on intact membrane and on Nonidet P-40 solubilized membrane protein using antitubulin antibodies in antibody retention and radioimmune assays. Membrane tubulin content varied from 2.2 to 4.4 .mu.g/mg protein. The involvement of membrane tubulin in tubulin-membrane interactions remains questionable since erythrocyte membranes devoid of membrane tubulin exhibited a low (1/10 of that of rat liver plasma membranes) but significant tubulin-binding activity. Membranes specialized in different cell functions possess high-affinity, large-capacity tubulin-binding sites. Tubulin-membrane interaction may involve the assembly of a limited number of tubulin dimers in a micropolymerization process initiated by a membrane component present in various cellular membranes or by different membrane components exhibiting a common property.