Reversible association of a 97-kDa protein complex found at the tips of ciliary microtubules with in vitro assembled microtubules.
Using a human autoimmune CREST antiserum, we identified a 97-kDa polypeptide in Tetrahymena cilia at the plus ends of ciliary microtubules and mammalian kinetochores (Miller, J. M., Wang, W., Balczon, R., and Dentler, W. L. (1990) J. Cell Biol. 110, 703-714). In this study, we examined the interactions of the ciliary 97-kDa protein with microtubules assembled from purified bovine brain tubulin. The 97-kDa protein binds to microtubules and is released from them with 75 mM MgCl2, the same condition used to release it from ciliary microtubules. The 97-kDa protein-microtubule association can be disrupted by ATP and by adenosine 5'-O-(thio-triphosphate), and this ATP sensitivity requires a soluble factor in the crude 97-kDa protein fraction. Fractionation of the crude 97-kDa protein fractions by gel filtration chromatography or by sucrose gradient centrifugation causes the loss of the microtubule binding activity of the 97-kDa protein. Fractions containing a high molecular weight protein complex (molecular mass, approximately 1500-2000 kDa) from the column or gradient fractions can restore the microtubule binding ability of the 97-kDa protein. These results suggest that the 97-kDa protein is part of a high molecular weight protein complex and that the complex, not the 97-kDa protein alone, is required for the 97-kDa protein-microtubule association. The association of the 97-kDa protein with microtubules is sensitive to ATP, which suggests that the 97-kDa protein-microtubule interaction may be regulated by an ATPase(s) or protein kinase(s)/phosphatase(s).
Wang
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W
Department of Physiology and Cell Biology, University of Kansas, Lawrence 66045.
Suprenant
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Dentler
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WL
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D002923:Cilia / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008868:Microtubule Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D015800:Protozoan Proteins / Q000378:metabolism; D016808:Tetrahymena thermophila / Q000378:metabolism
D008868:Microtubule Proteins; D015800:Protozoan Proteins; D000255:Adenosine Triphosphate