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J Cell Biol. 1992 September 1; 118(5): 1177–1188.
PMCID: PMC2289586

The motile beta/IC1 subunit of sea urchin sperm outer arm dynein does not form a rigor bond


We used in vitro translocation and cosedimentation assays to study the microtubule binding properties of sea urchin sperm outer arm dynein and its beta/IC1 subunit. Microtubules glided on glass-absorbed sea urchin dynein for a period of time directly proportional to the initial MgATP2- concentration and then detached when 70-95% of the MgATP2- was hydrolyzed. Detachment resulted from MgATP2- depletion, because (a) perfusion with fresh buffer containing MgATP2- reconstituted binding and gliding, (b) microtubules glided many minutes with an ATP- regenerating system at ATP concentrations which alone supported gliding for only 1-2 min, and (c) microtubules detached upon total hydrolysis of ATP by an ATP-removal system. The products of ATP hydrolysis antagonized binding and gliding; as little as a threefold excess of ADP/Pi over ATP resulted in complete loss of microtubule binding and translocation by the beta/IC1 subunit. In contrast to the situation with sea urchin dynein, microtubules ceased gliding but remained bound to glass-absorbed Tetrahymena outer arm dynein when MgATP2- was exhausted. Cosedimentation assays showed that Tetrahymena outer arm dynein sedimented with microtubules in an ATP-sensitive manner, as previously reported (Porter, M.E., and K. A. Johnson. J. Biol. Chem. 258: 6575-6581). However, the beta/IC1 subunit of sea urchin dynein did not cosediment with microtubules in the absence of ATP. Thus, this subunit, while capable of generating motility, lacks both structural and rigor-type microtubule binding.

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Selected References

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  • Bell CW, Gibbons IR. Structure of the dynein-1 outer arm in sea urchin sperm flagella. II. Analysis by proteolytic cleavage. J Biol Chem. 1982 Jan 10;257(1):516–522. [PubMed]
  • Bradford MM. 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. [PubMed]
  • Brokaw CJ. Future directions for studies of mechanisms for generating flagellar bending waves. J Cell Sci Suppl. 1986;4:103–113. [PubMed]
  • Fox LA, Sale WS. Direction of force generated by the inner row of dynein arms on flagellar microtubules. J Cell Biol. 1987 Oct;105(4):1781–1787. [PMC free article] [PubMed]
  • Gatti JL, King SM, Moss AG, Witman GB. Outer arm dynein from trout spermatozoa. Purification, polypeptide composition, and enzymatic properties. J Biol Chem. 1989 Jul 5;264(19):11450–11457. [PubMed]
  • Gibbons IR. The molecular basis of flagellar motility in sea urchin spermatozoa. Soc Gen Physiol Ser. 1975;30:207–232. [PubMed]
  • Gibbons BH, Gibbons IR. Properties of flagellar "rigor waves" formed by abrupt removal of adenosine triphosphate from actively swimming sea urchin sperm. J Cell Biol. 1974 Dec;63(3):970–985. [PMC free article] [PubMed]
  • Gibbons BH, Gibbons IR. Relationship between the latent adenosine triphosphatase state of dynein 1 and its ability to recombine functionally with KCl-extracted sea urchin sperm flagella. J Biol Chem. 1979 Jan 10;254(1):197–201. [PubMed]
  • Gibbons BH, Tang WJ, Gibbons IR. Organic anions stabilize the reactivated motility of sperm flagella and the latency of dynein 1 ATPase activity. J Cell Biol. 1985 Oct;101(4):1281–1287. [PMC free article] [PubMed]
  • Gibbons IR, Fronk E. A latent adenosine triphosphatase form of dynein 1 from sea urchin sperm flagella. J Biol Chem. 1979 Jan 10;254(1):187–196. [PubMed]
  • Gibbons IR, Gibbons BH, Mocz G, Asai DJ. Multiple nucleotide-binding sites in the sequence of dynein beta heavy chain. Nature. 1991 Aug 15;352(6336):640–643. [PubMed]
  • Holzbaur EL, Johnson KA. ADP release is rate limiting in steady-state turnover by the dynein adenosinetriphosphatase. Biochemistry. 1989 Jun 27;28(13):5577–5585. [PubMed]
  • Ishijima S, Witman GB. Flagellar movement of intact and demembranated, reactivated ram spermatozoa. Cell Motil Cytoskeleton. 1987;8(4):375–391. [PubMed]
  • Johnson KA. The pathway of ATP hydrolysis by dynein. Kinetics of a presteady state phosphate burst. J Biol Chem. 1983 Nov 25;258(22):13825–13832. [PubMed]
  • Johnson KA. Pathway of the microtubule-dynein ATPase and the structure of dynein: a comparison with actomyosin. Annu Rev Biophys Biophys Chem. 1985;14:161–188. [PubMed]
  • Johnson KA. Preparation and properties of dynein from Tetrahymena cilia. Methods Enzymol. 1986;134:306–317. [PubMed]
  • King SM, Witman GB. 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]
  • King SM, Witman GB. 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]
  • King SM, Gatti JL, Moss AG, Witman GB. Outer-arm dynein from trout spermatozoa: substructural organization. Cell Motil Cytoskeleton. 1990;16(4):266–278. [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Lee-Eiford A, Ow RA, Gibbons IR. Specific cleavage of dynein heavy chains by ultraviolet irradiation in the presence of ATP and vanadate. J Biol Chem. 1986 Feb 15;261(5):2337–2342. [PubMed]
  • Mitchell DR, Warner FD. Interactions of dynein arms with b subfibers of Tetrahymena cilia: quantitation of the effects of magnesium and adenosine triphosphate. J Cell Biol. 1980 Oct;87(1):84–97. [PMC free article] [PubMed]
  • Mitchell DR, Warner FD. Binding of dynein 21 S ATPase to microtubules. Effects of ionic conditions and substrate analogs. J Biol Chem. 1981 Dec 10;256(23):12535–12544. [PubMed]
  • Moss AG, Sale WS, Fox LA, Witman GB. The alpha subunit of sea urchin sperm outer arm dynein mediates structural and rigor binding to microtubules. J Cell Biol. 1992 Sep;118(5):1189–1200. [PMC free article] [PubMed]
  • Ogawa K. Four ATP-binding sites in the midregion of the beta heavy chain of dynein. Nature. 1991 Aug 15;352(6336):643–645. [PubMed]
  • Okuno M. Inhibition and relaxation of sea urchin sperm flagella by vanadate. J Cell Biol. 1980 Jun;85(3):712–725. [PMC free article] [PubMed]
  • Okuno M, Brokaw CJ. Inhibition of movement of trition-demembranated sea-urchin sperm flagella by Mg2+, ATP4-, ADP and P1. J Cell Sci. 1979 Aug;38:105–123. [PubMed]
  • Omoto CK, Johnson KA. Activation of the dynein adenosinetriphosphatase by microtubules. Biochemistry. 1986 Jan 28;25(2):419–427. [PubMed]
  • Paschal BM, King SM, Moss AG, Collins CA, Vallee RB, Witman GB. Isolated flagellar outer arm dynein translocates brain microtubules in vitro. Nature. 1987 Dec 17;330(6149):672–674. [PubMed]
  • Penningroth SM, Witman GB. Effects of adenylyl imidodiphosphate, a nonhydrolyzable adenosine triphosphate analog, on reactivated and rigor wave sea urchin sperm. J Cell Biol. 1978 Dec;79(3):827–832. [PMC free article] [PubMed]
  • Penningroth SM, Olehnik K, Cheung A. ATP formation from adenyl-5'-yl imidodiphosphate, a nonhydrolyzable ATP analog. J Biol Chem. 1980 Oct 25;255(20):9545–9548. [PubMed]
  • Pfister KK, Witman GB. Subfractionation of Chlamydomonas 18 S dynein into two unique subunits containing ATPase activity. J Biol Chem. 1984 Oct 10;259(19):12072–12080. [PubMed]
  • Pfister KK, Haley BE, Witman GB. The photoaffinity probe 8-azidoadenosine 5'-triphosphate selectively labels the heavy chain of Chlamydomonas 12 S dynein. J Biol Chem. 1984 Jul 10;259(13):8499–8504. [PubMed]
  • Porter ME, Johnson KA. 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]
  • Porter ME, Johnson KA. Transient state kinetic analysis of the ATP-induced dissociation of the dynein-microtubule complex. J Biol Chem. 1983 May 25;258(10):6582–6587. [PubMed]
  • Porter ME, Johnson KA. Dynein structure and function. Annu Rev Cell Biol. 1989;5:119–151. [PubMed]
  • Sale WS, Fox LA. Isolated beta-heavy chain subunit of dynein translocates microtubules in vitro. J Cell Biol. 1988 Nov;107(5):1793–1797. [PMC free article] [PubMed]
  • Sale WS, Satir P. Direction of active sliding of microtubules in Tetrahymena cilia. Proc Natl Acad Sci U S A. 1977 May;74(5):2045–2049. [PubMed]
  • Satir P. Studies on cilia. 3. Further studies on the cilium tip and a "sliding filament" model of ciliary motility. J Cell Biol. 1968 Oct;39(1):77–94. [PMC free article] [PubMed]
  • Satir P. Mechanisms and controls of microtubule sliding in cilia. Symp Soc Exp Biol. 1982;35:179–201. [PubMed]
  • Shimizu T, Johnson KA. Kinetic evidence for multiple dynein ATPase sites. J Biol Chem. 1983 Nov 25;258(22):13841–13846. [PubMed]
  • Shimizu T, Furusawa K, Ohashi S, Toyoshima YY, Okuno M, Malik F, Vale RD. Nucleotide specificity of the enzymatic and motile activities of dynein, kinesin, and heavy meromyosin. J Cell Biol. 1991 Mar;112(6):1189–1197. [PMC free article] [PubMed]
  • Shingyoji C, Murakami A, Takahashi K. Local reactivation of Triton-extracted flagella by iontophoretic application of ATP. Nature. 1977 Jan 20;265(5591):269–270. [PubMed]
  • Spungin B, Avolio J, Arden S, Satir P. Dynein arm attachment probed with a non-hydrolyzable ATP analog. Structural evidence for patterns of activity. J Mol Biol. 1987 Oct 20;197(4):671–677. [PubMed]
  • Summers KE, Gibbons IR. Adenosine triphosphate-induced sliding of tubules in trypsin-treated flagella of sea-urchin sperm. Proc Natl Acad Sci U S A. 1971 Dec;68(12):3092–3096. [PubMed]
  • Takahashi M, Tonomura Y. Binding of 30s dynein with the B-tubule of the outer doublet of axonemes from Tetrahymena pyriformis and adenosine triphosphate-induced dissociation of the complex. J Biochem. 1978 Dec;84(6):1339–1355. [PubMed]
  • Tang WJ, Bell CW, Sale WS, Gibbons IR. 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]
  • Toyoshima YY. Chymotryptic digestion of Tetrahymena 22S dynein. I. Decomposition of three-headed 22S dynein to one- and two-headed particles. J Cell Biol. 1987 Aug;105(2):887–895. [PMC free article] [PubMed]
  • Toyoshima YY. Chymotryptic digestion of Tetrahymena ciliary dynein. II. Pathway of the degradation of 22S dynein heavy chains. J Cell Biol. 1987 Aug;105(2):897–901. [PMC free article] [PubMed]
  • Vale RD, Toyoshima YY. Rotation and translocation of microtubules in vitro induced by dyneins from Tetrahymena cilia. Cell. 1988 Feb 12;52(3):459–469. [PubMed]
  • Vale RD, Toyoshima YY. Microtubule translocation properties of intact and proteolytically digested dyneins from Tetrahymena cilia. J Cell Biol. 1989 Jun;108(6):2327–2334. [PMC free article] [PubMed]
  • Vale RD, Soll DR, Gibbons IR. One-dimensional diffusion of microtubules bound to flagellar dynein. Cell. 1989 Dec 1;59(5):915–925. [PubMed]
  • Vallee RB. Reversible assembly purification of microtubules without assembly-promoting agents and further purification of tubulin, microtubule-associated proteins, and MAP fragments. Methods Enzymol. 1986;134:89–104. [PubMed]
  • Wais-Steider J, Satir P. Effect of vanadate on gill cilia: switching mechanism in ciliary beat. J Supramol Struct. 1979;11(3):339–347. [PubMed]
  • Warner FD. Cation-induced attachment of ciliary dynein cross-bridges. J Cell Biol. 1978 Jun;77(3):R19–R26. [PMC free article] [PubMed]
  • Warner FD, McIlvain JH. Binding stoichiometry of 21S dynein to A and B subfiber microtubules. Cell Motil. 1982;2(5):429–443. [PubMed]
  • Warner FD, Perreault JG, McIlvain JH. Rebinding of Tetrahymena 13 S and 21 S dynein ATPases to extracted doublet microtubules. The inner row and outer row dynein arms. J Cell Sci. 1985 Aug;77:263–287. [PubMed]
  • Witman GB. Axonemal dyneins. Curr Opin Cell Biol. 1992 Feb;4(1):74–79. [PubMed]

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