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.
Severin
F F
FF
Institute of Protein Research, Academy of Sciences of the USSR, Pushchino, Moscow.
Shanina
N A
NA
Kuznetsov
S A
SA
Gelfand
V I
VI
D000255:Adenosine Triphosphate / Q000378:metabolism; D000313:Adrenal Medulla / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002499:Centrifugation, Density Gradient; D002837:Chromaffin Granules / Q000378:metabolism* / Q000648:ultrastructure; D002846:Chromatography, Affinity; D008869:Microtubule-Associated Proteins / Q000378:metabolism* / Q000648:ultrastructure; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure; D014357:Trypsin / Q000378:metabolism
D008869:Microtubule-Associated Proteins; D000255:Adenosine Triphosphate; D014357:Trypsin