MAP2-mediated binding of chromaffin granules to microtubules.

We have examined the interaction of chromaffin granules from bovine adrenal medulla with microtubules. Chromaffin granules were mixed with microtubules made of phosphocellulose-purified tubulin, and pelleted through a 1.6 M sucrose cushion at 12,000 x g for 10 min. Both components (granules and microtubules) were pelleted when added together but not separately. This result indicates that granules form a heavy complex with the microtubules. Such a complex was visualized by an electron microscopy of the granule/microtubule mixture. Treatment of the granules with trypsin abolished their ability to interact with the microtubules. The binding of the granules to the microtubules; (i) was not sensitive to ATP; and (ii) was completely inhibited by the cleavage of C-terminal peptides of alpha- and beta-subunits of tubulin with subtilisin. These relationships suggest that the granule binding is mediated by one of the structural microtubule-associated proteins rather than by microtubule-dependent translocators. For identification of protein(s) mediating the binding, the granules were solubilized with Triton X-100, soluble proteins were mixed with the microtubules, and microtubules with bound proteins were pelleted through a glycerol cushion. At least one granule protein interacting with the microtubules was found in the pellet. This protein was identified as MAP2 according to its electrophoretic mobility and reactivity with a MAP2 antibody. Affinity chromatography of solubilized proteins on a column containing taxol-stabilized microtubules also revealed MAP2 as a protein of chromaffin granules interacting with the microtubules.