The Mr 78,000 intermediate chain of Chlamydomonas outer arm dynein interacts with alpha-tubulin in situ.
We have used the zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to examine protein-protein associations within purified outer arm dynein and axonemes from Chlamydomonas flagella. When axonemes were treated with 0.5-1 mM EDC in either the presence or absence of ATP/vanadate, a polypeptide band of Mr 127,000 recognized by monoclonal antibody 1878A (specific for the Mr 78,000 intermediate chain (IC78) of outer arm dynein) was generated. This conjugate was not obtained when purified dynein was treated with EDC. Further immunological analysis demonstrated that this complex also contained alpha- (but not beta-) tubulin. These results indicate that IC78 interacts with alpha-tubulin in situ in an ATP-insensitive manner. Identification of this interface between dynein and tubulin suggests that IC78, which probably is located at the base of the dynein particle (King, S. M., and Witman, G. B. (1990) J. Biol. Chem. 265, 19807-19811), contributes to the structural attachment of the dynein arms to the A-tubules of the outer doublet microtubules. Analysis of the cross-linked products from the purified dynein revealed several additional interactions involving the intermediate chains; these adducts provide further evidence for an intermediate chain/light chain complex within dynein and confirm that IC78 and IC69 associate directly.
King
S M
SM
Cell Biology Group, Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545.
Wilkerson
C G
CG
Witman
G B
GB
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000276:immunology; D002696:Chlamydomonas / Q000378:metabolism*; D003432:Cross-Linking Reagents; D004398:Dyneins / Q000276:immunology / Q000378:metabolism*; D005022:Ethyldimethylaminopropyl Carbodiimide; D015394:Molecular Structure; D011233:Precipitin Tests; D011485:Protein Binding; D014404:Tubulin / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D014404:Tubulin; D014638:Vanadates; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D004398:Dyneins; D005022:Ethyldimethylaminopropyl Carbodiimide