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. 1994 Aug 1;126(3):737–745. doi: 10.1083/jcb.126.3.737

Functional reconstitution of Chlamydomonas outer dynein arms from alpha- beta and gamma subunits: requirement of a third factor

PMCID: PMC2120151  PMID: 8045937

Abstract

The outer dynein arm of Chlamydomonas flagella, when isolated under Mg(2+)-free conditions, tends to dissociate into an 11 to 12S particle (12S dynein) containing the gamma heavy chain and a 21S particle (called 18S dynein) containing the alpha and beta heavy chains. We show here that functional outer arms can be reconstituted by the addition of 12S and 18S dyneins to the axonemes of the outer armless mutants oda1- oda6. A third factor that sediments at integral 7S is required for efficient reconstitution of the outer arms on the axonemes of oda1 and oda3. However, this factor is not necessary for reconstitution on the axonemes of oda2, oda4, oda5, and oda6. SDS-PAGE analysis indicates that the axonemes of the former two mutants lack a integral of 70-kD polypeptide that is present in those of the other mutants as well as in the 7S fraction from the wild-type extract. Furthermore, electron micrographs of axonemal cross sections revealed that the latter four mutants, but not oda1 or oda3, have small pointed structures on the outer doublets, at a position in cross section where outer arms normally occur. We suggest that the 7S factor constitutes the pointed structure on the outer doublets and facilitates attachment of the outer arm. The discovery of this structure raises a new question as to how the attachment site for the outer arm dynein is determined within the axoneme.

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Selected References

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  1. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  2. Goodenough U., Heuser J. Structural comparison of purified dynein proteins with in situ dynein arms. J Mol Biol. 1984 Dec 25;180(4):1083–1118. doi: 10.1016/0022-2836(84)90272-9. [DOI] [PubMed] [Google Scholar]
  3. Gorman D. S., Levine R. P. Cytochrome f and plastocyanin: their sequence in the photosynthetic electron transport chain of Chlamydomonas reinhardi. Proc Natl Acad Sci U S A. 1965 Dec;54(6):1665–1669. doi: 10.1073/pnas.54.6.1665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Hoops H. J., Witman G. B. Outer doublet heterogeneity reveals structural polarity related to beat direction in Chlamydomonas flagella. J Cell Biol. 1983 Sep;97(3):902–908. doi: 10.1083/jcb.97.3.902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Jarvik J. W., Rosenbaum J. L. Oversized flagellar membrane protein in paralyzed mutants of Chlamydomonas reinhardrii. J Cell Biol. 1980 May;85(2):258–272. doi: 10.1083/jcb.85.2.258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Kamiya R., Hasegawa E. Intrinsic difference in beat frequency between the two flagella of Chlamydomonas reinhardtii. Exp Cell Res. 1987 Nov;173(1):299–304. doi: 10.1016/0014-4827(87)90357-0. [DOI] [PubMed] [Google Scholar]
  7. Kamiya R. Mutations at twelve independent loci result in absence of outer dynein arms in Chylamydomonas reinhardtii. J Cell Biol. 1988 Dec;107(6 Pt 1):2253–2258. doi: 10.1083/jcb.107.6.2253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kamiya R., Witman G. B. Submicromolar levels of calcium control the balance of beating between the two flagella in demembranated models of Chlamydomonas. J Cell Biol. 1984 Jan;98(1):97–107. doi: 10.1083/jcb.98.1.97. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. King S. M., Wilkerson C. G., Witman G. B. The Mr 78,000 intermediate chain of Chlamydomonas outer arm dynein interacts with alpha-tubulin in situ. J Biol Chem. 1991 May 5;266(13):8401–8407. [PubMed] [Google Scholar]
  10. King S. M., Witman G. B. Localization of an intermediate chain of outer arm dynein by immunoelectron microscopy. J Biol Chem. 1990 Nov 15;265(32):19807–19811. [PubMed] [Google Scholar]
  11. King S. M., Witman G. B. Structure of the alpha and beta heavy chains of the outer arm dynein from Chlamydomonas flagella. Location of epitopes and protease-sensitive sites. J Biol Chem. 1988 Jul 5;263(19):9244–9255. [PubMed] [Google Scholar]
  12. King S. M., Witman G. B. Structure of the gamma heavy chain of the outer arm dynein from Chlamydomonas flagella. J Cell Biol. 1988 Nov;107(5):1799–1808. doi: 10.1083/jcb.107.5.1799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Mitchell D. R., Rosenbaum J. L. Protein-protein interactions in the 18S ATPase of Chlamydomonas outer dynein arms. Cell Motil Cytoskeleton. 1986;6(5):510–520. doi: 10.1002/cm.970060510. [DOI] [PubMed] [Google Scholar]
  14. Pfister K. K., Fay R. B., Witman G. B. Purification and polypeptide composition of dynein ATPases from Chlamydomonas flagella. Cell Motil. 1982;2(6):525–547. doi: 10.1002/cm.970020604. [DOI] [PubMed] [Google Scholar]
  15. Pfister K. K., Witman G. B. Subfractionation of Chlamydomonas 18 S dynein into two unique subunits containing ATPase activity. J Biol Chem. 1984 Oct 10;259(19):12072–12080. [PubMed] [Google Scholar]
  16. Piperno G., Luck D. J. Axonemal adenosine triphosphatases from flagella of Chlamydomonas reinhardtii. Purification of two dyneins. J Biol Chem. 1979 Apr 25;254(8):3084–3090. [PubMed] [Google Scholar]
  17. Piperno G., Ramanis Z., Smith E. F., Sale W. S. Three distinct inner dynein arms in Chlamydomonas flagella: molecular composition and location in the axoneme. J Cell Biol. 1990 Feb;110(2):379–389. doi: 10.1083/jcb.110.2.379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Porter M. E., Johnson K. A. Dynein structure and function. Annu Rev Cell Biol. 1989;5:119–151. doi: 10.1146/annurev.cb.05.110189.001003. [DOI] [PubMed] [Google Scholar]
  19. Sakakibara H., Mitchell D. R., Kamiya R. A Chlamydomonas outer arm dynein mutant missing the alpha heavy chain. J Cell Biol. 1991 May;113(3):615–622. doi: 10.1083/jcb.113.3.615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Sakakibara H., Takada S., King S. M., Witman G. B., Kamiya R. A Chlamydomonas outer arm dynein mutant with a truncated beta heavy chain. J Cell Biol. 1993 Aug;122(3):653–661. doi: 10.1083/jcb.122.3.653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Takada S., Sakakibara H., Kamiya R. Three-headed outer arm dynein from Chlamydomonas that can functionally combine with outer-arm-missing axonemes. J Biochem. 1992 Jun;111(6):758–762. doi: 10.1093/oxfordjournals.jbchem.a123832. [DOI] [PubMed] [Google Scholar]
  22. Tang W. J., Bell C. W., Sale W. S., Gibbons I. R. Structure of the dynein-1 outer arm in sea urchin sperm flagella. I. Analysis by separation of subunits. J Biol Chem. 1982 Jan 10;257(1):508–515. [PubMed] [Google Scholar]
  23. Witman G. B. Isolation of Chlamydomonas flagella and flagellar axonemes. Methods Enzymol. 1986;134:280–290. doi: 10.1016/0076-6879(86)34096-5. [DOI] [PubMed] [Google Scholar]

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