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. 1994 Sep 2;126(6):1495–1507. doi: 10.1083/jcb.126.6.1495

Mutations in the SUP-PF-1 locus of Chlamydomonas reinhardtii identify a regulatory domain in the beta-dynein heavy chain

PMCID: PMC2290962  PMID: 8089181

Abstract

We have characterized a group of regulatory mutations that alter the activity of the outer dynein arms. Three mutations were obtained as suppressors of the paralyzed central pair mutant pf6 (Luck, D.J.L., and G. Piperno. 1989. Cell Movement. pp. 49-60), whereas two others were obtained as suppressors of the central pair mutant pfl6. Recombination analysis and complementation tests indicate that all five mutations are alleles at the SUP-PF-1/ODA4 locus and that each allele can restore motility to radial spoke and central pair defective strains. Restriction fragment length polymorphism analysis with a genomic probe for the beta-dynein heavy chain (DHC) gene confirms that this locus is tightly linked to the beta-DHC gene. Although all five mutant sup-pf-1 alleles alter the activity of the outer dynein arm as assayed by measurements of flagellar motility, only two alleles have a discernable polypeptide defect by SDS-PAGE. We have used photolytic and proteolytic cleavage procedures to localize the polypeptide defect to an approximately 100-kD domain downstream from the last putative nucleotide binding site. This region is encoded by approximately 5 kb of genomic DNA (Mitchell, D.R., and K. Brown. 1994. J. Cell Sci. 107:653-644). PCR amplification of wild-type and mutant DNA across this region identified one PCR product that was consistently smaller in the sup-pf-1 DNA. Direct DNA sequencing of the PCR products revealed that two of the sup-pf-1 mutations are distinct, in-frame deletions. These deletions occur within a region that is predicted to encode a small alpha-helical coiled-coil domain of the beta-DHC. This domain may play a role in protein-protein interactions within the outer dynein arm. Since both the size and location of this domain have been conserved in all axonemal and cytoplasmic DHCs sequenced to date, it presumably performs a common function in all dynein isoforms.

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Selected References

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  1. Asai D. J., Brokaw C. J. Dynein heavy chain isoforms and axonemal motility. Trends Cell Biol. 1993 Nov;3(11):398–402. doi: 10.1016/0962-8924(93)90090-n. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Brokaw C. J., Kamiya R. Bending patterns of Chlamydomonas flagella: IV. Mutants with defects in inner and outer dynein arms indicate differences in dynein arm function. Cell Motil Cytoskeleton. 1987;8(1):68–75. doi: 10.1002/cm.970080110. [DOI] [PubMed] [Google Scholar]
  4. Brokaw C. J., Luck D. J., Huang B. Analysis of the movement of Chlamydomonas flagella:" the function of the radial-spoke system is revealed by comparison of wild-type and mutant flagella. J Cell Biol. 1982 Mar;92(3):722–732. doi: 10.1083/jcb.92.3.722. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chandra R., Endow S. A., Salmon E. D. An N-terminal truncation of the ncd motor protein supports diffusional movement of microtubules in motility assays. J Cell Sci. 1993 Mar;104(Pt 3):899–906. doi: 10.1242/jcs.104.3.899. [DOI] [PubMed] [Google Scholar]
  6. Chandra R., Salmon E. D., Erickson H. P., Lockhart A., Endow S. A. Structural and functional domains of the Drosophila ncd microtubule motor protein. J Biol Chem. 1993 Apr 25;268(12):9005–9013. [PubMed] [Google Scholar]
  7. Chou P. Y., Fasman G. D. Empirical predictions of protein conformation. Annu Rev Biochem. 1978;47:251–276. doi: 10.1146/annurev.bi.47.070178.001343. [DOI] [PubMed] [Google Scholar]
  8. Collier V. L., Kronert W. A., O'Donnell P. T., Edwards K. A., Bernstein S. I. Alternative myosin hinge regions are utilized in a tissue-specific fashion that correlates with muscle contraction speed. Genes Dev. 1990 Jun;4(6):885–895. doi: 10.1101/gad.4.6.885. [DOI] [PubMed] [Google Scholar]
  9. Dutcher S. K., Huang B., Luck D. J. Genetic dissection of the central pair microtubules of the flagella of Chlamydomonas reinhardtii. J Cell Biol. 1984 Jan;98(1):229–236. doi: 10.1083/jcb.98.1.229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ebersold W. T. Chlamydomonas reinhardi: heterozygous diploid strains. Science. 1967 Jul 28;157(3787):447–449. doi: 10.1126/science.157.3787.447. [DOI] [PubMed] [Google Scholar]
  11. Ferris P. J., Goodenough U. W. The mating-type locus of Chlamydomonas reinhardtii contains highly rearranged DNA sequences. Cell. 1994 Mar 25;76(6):1135–1145. doi: 10.1016/0092-8674(94)90389-1. [DOI] [PubMed] [Google Scholar]
  12. Garnier J., Osguthorpe D. J., Robson B. Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J Mol Biol. 1978 Mar 25;120(1):97–120. doi: 10.1016/0022-2836(78)90297-8. [DOI] [PubMed] [Google Scholar]
  13. Gibbons I. R., Gibbons B. H., Mocz G., Asai D. J. Multiple nucleotide-binding sites in the sequence of dynein beta heavy chain. Nature. 1991 Aug 15;352(6336):640–643. doi: 10.1038/352640a0. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Huang B., Ramanis Z., Luck D. J. Suppressor mutations in Chlamydomonas reveal a regulatory mechanism for Flagellar function. Cell. 1982 Jan;28(1):115–124. doi: 10.1016/0092-8674(82)90381-6. [DOI] [PubMed] [Google Scholar]
  16. Johnson D. E., Dutcher S. K. Molecular studies of linkage group XIX of Chlamydomonas reinhardtii: evidence against a basal body location. J Cell Biol. 1991 Apr;113(2):339–346. doi: 10.1083/jcb.113.2.339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. King S. M., Otter T., Witman G. B. Purification and characterization of Chlamydomonas flagellar dyneins. Methods Enzymol. 1986;134:291–306. doi: 10.1016/0076-6879(86)34097-7. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. King S. M., Witman G. B. Structure of the alpha and beta heavy chains of the outer arm dynein from Chlamydomonas flagella. Masses of chains and sites of ultraviolet-induced vanadate-dependent cleavage. J Biol Chem. 1987 Dec 25;262(36):17596–17604. [PubMed] [Google Scholar]
  22. Kurimoto E., Kamiya R. Microtubule sliding in flagellar axonemes of Chlamydomonas mutants missing inner- or outer-arm dynein: velocity measurements on new types of mutants by an improved method. Cell Motil Cytoskeleton. 1991;19(4):275–281. doi: 10.1002/cm.970190406. [DOI] [PubMed] [Google Scholar]
  23. LEVINE R. P., EBERSOLD W. T. The genetics and cytology of Chlamydomonas. Annu Rev Microbiol. 1960;14:197–216. doi: 10.1146/annurev.mi.14.100160.001213. [DOI] [PubMed] [Google Scholar]
  24. Li M., McGrail M., Serr M., Hays T. S. Drosophila cytoplasmic dynein, a microtubule motor that is asymmetrically localized in the oocyte. J Cell Biol. 1994 Sep;126(6):1475–1494. doi: 10.1083/jcb.126.6.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Luck D., Piperno G., Ramanis Z., Huang B. Flagellar mutants of Chlamydomonas: studies of radial spoke-defective strains by dikaryon and revertant analysis. Proc Natl Acad Sci U S A. 1977 Aug;74(8):3456–3460. doi: 10.1073/pnas.74.8.3456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lupas A., Van Dyke M., Stock J. Predicting coiled coils from protein sequences. Science. 1991 May 24;252(5009):1162–1164. doi: 10.1126/science.252.5009.1162. [DOI] [PubMed] [Google Scholar]
  27. Mastronarde D. N., O'Toole E. T., McDonald K. L., McIntosh J. R., Porter M. E. Arrangement of inner dynein arms in wild-type and mutant flagella of Chlamydomonas. J Cell Biol. 1992 Sep;118(5):1145–1162. doi: 10.1083/jcb.118.5.1145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mikami A., Paschal B. M., Mazumdar M., Vallee R. B. Molecular cloning of the retrograde transport motor cytoplasmic dynein (MAP 1C). Neuron. 1993 May;10(5):787–796. doi: 10.1016/0896-6273(93)90195-w. [DOI] [PubMed] [Google Scholar]
  29. Mitchell D. R., Brown K. S. Sequence analysis of the Chlamydomonas alpha and beta dynein heavy chain genes. J Cell Sci. 1994 Mar;107(Pt 3):635–644. doi: 10.1242/jcs.107.3.635. [DOI] [PubMed] [Google Scholar]
  30. Mitchell D. R., Kang Y. Identification of oda6 as a Chlamydomonas dynein mutant by rescue with the wild-type gene. J Cell Biol. 1991 May;113(4):835–842. doi: 10.1083/jcb.113.4.835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Mitchell D. R., Kang Y. Reversion analysis of dynein intermediate chain function. J Cell Sci. 1993 Aug;105(Pt 4):1069–1078. doi: 10.1242/jcs.105.4.1069. [DOI] [PubMed] [Google Scholar]
  32. Mitchell D. R., Rosenbaum J. L. A motile Chlamydomonas flagellar mutant that lacks outer dynein arms. J Cell Biol. 1985 Apr;100(4):1228–1234. doi: 10.1083/jcb.100.4.1228. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Mocz G., Gibbons I. R. ATP-insensitive interaction of the amino-terminal region of the beta heavy chain of dynein with microtubules. Biochemistry. 1993 Apr 6;32(13):3456–3460. doi: 10.1021/bi00064a032. [DOI] [PubMed] [Google Scholar]
  34. Neville D. M., Jr Molecular weight determination of protein-dodecyl sulfate complexes by gel electrophoresis in a discontinuous buffer system. J Biol Chem. 1971 Oct 25;246(20):6328–6334. [PubMed] [Google Scholar]
  35. Ogawa K. Four ATP-binding sites in the midregion of the beta heavy chain of dynein. Nature. 1991 Aug 15;352(6336):643–645. doi: 10.1038/352643a0. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Piperno G., Mead K., Shestak W. The inner dynein arms I2 interact with a "dynein regulatory complex" in Chlamydomonas flagella. J Cell Biol. 1992 Sep;118(6):1455–1463. doi: 10.1083/jcb.118.6.1455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Porter M. E., Johnson K. A. Characterization of the ATP-sensitive binding of Tetrahymena 30 S dynein to bovine brain microtubules. J Biol Chem. 1983 May 25;258(10):6575–6581. [PubMed] [Google Scholar]
  39. 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]
  40. Porter M. E., Power J., Dutcher S. K. Extragenic suppressors of paralyzed flagellar mutations in Chlamydomonas reinhardtii identify loci that alter the inner dynein arms. J Cell Biol. 1992 Sep;118(5):1163–1176. doi: 10.1083/jcb.118.5.1163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Porter M. E., Scholey J. M., Stemple D. L., Vigers G. P., Vale R. D., Sheetz M. P., McIntosh J. R. Characterization of the microtubule movement produced by sea urchin egg kinesin. J Biol Chem. 1987 Feb 25;262(6):2794–2802. [PubMed] [Google Scholar]
  42. Ranum L. P., Thompson M. D., Schloss J. A., Lefebvre P. A., Silflow C. D. Mapping flagellar genes in Chlamydomonas using restriction fragment length polymorphisms. Genetics. 1988 Sep;120(1):109–122. doi: 10.1093/genetics/120.1.109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. 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]
  45. Smith E. F., Sale W. S. Regulation of dynein-driven microtubule sliding by the radial spokes in flagella. Science. 1992 Sep 11;257(5076):1557–1559. doi: 10.1126/science.1387971. [DOI] [PubMed] [Google Scholar]
  46. Stewart R. J., Thaler J. P., Goldstein L. S. Direction of microtubule movement is an intrinsic property of the motor domains of kinesin heavy chain and Drosophila ncd protein. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):5209–5213. doi: 10.1073/pnas.90.11.5209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Uyeda T. Q., Spudich J. A. A functional recombinant myosin II lacking a regulatory light chain-binding site. Science. 1993 Dec 17;262(5141):1867–1870. doi: 10.1126/science.8266074. [DOI] [PubMed] [Google Scholar]
  48. Vale R. D., Toyoshima Y. Y. Rotation and translocation of microtubules in vitro induced by dyneins from Tetrahymena cilia. Cell. 1988 Feb 12;52(3):459–469. doi: 10.1016/s0092-8674(88)80038-2. [DOI] [PubMed] [Google Scholar]
  49. Vallee R. B., Wall J. S., Paschal B. M., Shpetner H. S. Microtubule-associated protein 1C from brain is a two-headed cytosolic dynein. Nature. 1988 Apr 7;332(6164):561–563. doi: 10.1038/332561a0. [DOI] [PubMed] [Google Scholar]
  50. Vallee R. Molecular analysis of the microtubule motor dynein. Proc Natl Acad Sci U S A. 1993 Oct 1;90(19):8769–8772. doi: 10.1073/pnas.90.19.8769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Waxman L., Goldberg A. L. Protease La from Escherichia coli hydrolyzes ATP and proteins in a linked fashion. Proc Natl Acad Sci U S A. 1982 Aug;79(16):4883–4887. doi: 10.1073/pnas.79.16.4883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wilkerson C. G., King S. M., Witman G. B. Molecular analysis of the gamma heavy chain of Chlamydomonas flagellar outer-arm dynein. J Cell Sci. 1994 Mar;107(Pt 3):497–506. doi: 10.1242/jcs.107.3.497. [DOI] [PubMed] [Google Scholar]
  53. Williams B. D., Mitchell D. R., Rosenbaum J. L. Molecular cloning and expression of flagellar radial spoke and dynein genes of Chlamydomonas. J Cell Biol. 1986 Jul;103(1):1–11. doi: 10.1083/jcb.103.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. 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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