MAP2‐mediated binding of chromaffin granules to microtubules

FEBS Letters

Kuznetsov, S.A.¹  Shanina, N.A.¹  Gelfand, V.I.²  Severin, F.F.² 

[1] Department of Molecular Biology, Moscow State University, Moscow 119899, USSR;Institute of Protein Research, Academy of Sciences of the USSR, Pushchino, Moscow Region, 142292 USSR

关键词: Chromaffin granule;    Microtubule;    Membrane-microtubule interaction;    MAP2;    Adrenal medulla;    S-microtubules;    microtubules made of subtilisindigested tubulin;    MAPs;    microtubule-associated proteins;    SDS-PAGE;    electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulfate;    TI;    soybean trypsin inhibitor;    EGTA;    [ethylenebis(oxyethylenenitrilo)tetraacetic acid;    BSA;    bovine serum albumin;   

DOI  :  10.1016/0014-5793(91)80445-9

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

We have examined the interaction of chromaffin granules from bovine adrenal modulla with microtubules. Chromaffin granules were mixed with microtubules made of phosphocellulose-purified tubulin, and pelleted through a 1.6 M sucrose cushion at 12000 × 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 α- and β-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.

【 授权许可】

Unknown