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J Cell Biol. 1995 October 2; 131(2): 399–409.
PMCID: PMC2199976

The 78,000-M(r) intermediate chain of Chlamydomonas outer arm dynein is a microtubule-binding protein

Abstract

A previous study (King et al., 1991. J. Biol. Chem. 266:8401-8407) showed that the 78,000-M(r) intermediate chain (IC78) from the Chlamydomonas outer arm dynein is in direct contact with alpha-tubulin in situ, suggesting that this protein may be involved in binding the dynein to the doublet microtubules. Molecular genetic analysis of this chain recently demonstrated that it is a WD repeat protein essential for outer arm assembly (Wilkerson et al., 1995.J. Cell Biol. 129:169- 178). We have now transcribed and translated IC78 in vitro, and demonstrate that this molecule binds axonemes and microtubules, whereas a homologous protein (the 69,000-M(r) intermediate chain [IC69] of Chlamydomonas outer arm dynein) does not. Thus, IC78 is a bona fide microtubule-binding protein. Taken together with the previous results, these findings indicate that IC78 is likely to provide at least some of the adhesive force that holds the dynein to the doublet microtubule, and support the general hypothesis that the dynein intermediate chains are involved in targeting different dyneins to the specific cell organelles with which they associate. Analysis of the binding activities of various IC78 deletion constructs translated in vitro identified discrete regions of IC78 that affected the binding to microtubules; two of these regions are specifically missing in IC69. Previous studies also showed that IC78 is in direct contact with IC69; the current work indicates that the region of IC78 that mediates this interaction is coincident with two of IC78's WD repeats. This supports the hypothesis that these repeats are involved in protein-protein interactions within the dynein complex.

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Selected References

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  • Adzhubei AA, Sternberg MJ. Conservation of polyproline II helices in homologous proteins: implications for structure prediction by model building. Protein Sci. 1994 Dec;3(12):2395–2410. [PubMed]
  • Aizawa H, Emori Y, Mori A, Murofushi H, Sakai H, Suzuki K. Functional analyses of the domain structure of microtubule-associated protein-4 (MAP-U). J Biol Chem. 1991 May 25;266(15):9841–9846. [PubMed]
  • 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]
  • Butner KA, Kirschner MW. Tau protein binds to microtubules through a flexible array of distributed weak sites. J Cell Biol. 1991 Nov;115(3):717–730. [PMC free article] [PubMed]
  • Chapin SJ, Bulinski JC. Non-neuronal 210 x 10(3) Mr microtubule-associated protein (MAP4) contains a domain homologous to the microtubule-binding domains of neuronal MAP2 and tau. J Cell Sci. 1991 Jan;98(Pt 1):27–36. [PubMed]
  • Devereux J, Haeberli P, Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. [PMC free article] [PubMed]
  • Dillman JF, 3rd, Pfister KK. Differential phosphorylation in vivo of cytoplasmic dynein associated with anterogradely moving organelles. J Cell Biol. 1994 Dec;127(6 Pt 1):1671–1681. [PMC free article] [PubMed]
  • 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. [PubMed]
  • Haimo LT, Fenton RD. Interaction of Chlamydomonas dynein with tubulin. Cell Motil Cytoskeleton. 1988;9(2):129–139. [PubMed]
  • Haimo LT, Telzer BR, Rosenbaum JL. Dynein binds to and crossbridges cytoplasmic microtubules. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5759–5763. [PubMed]
  • Hemphill A, Affolter M, Seebeck T. A novel microtubule-binding motif identified in a high molecular weight microtubule-associated protein from Trypanosoma brucei. J Cell Biol. 1992 Apr;117(1):95–103. [PMC free article] [PubMed]
  • Johnson KA, Wall JS. Structure and molecular weight of the dynein ATPase. J Cell Biol. 1983 Mar;96(3):669–678. [PMC free article] [PubMed]
  • King SM, Patel-King RS. The M(r) = 8,000 and 11,000 outer arm dynein light chains from Chlamydomonas flagella have cytoplasmic homologues. J Biol Chem. 1995 May 12;270(19):11445–11452. [PubMed]
  • King SM, Witman GB. Localization of an intermediate chain of outer arm dynein by immunoelectron microscopy. J Biol Chem. 1990 Nov 15;265(32):19807–19811. [PubMed]
  • King SM, Otter T, Witman GB. Characterization of monoclonal antibodies against Chlamydomonas flagellar dyneins by high-resolution protein blotting. Proc Natl Acad Sci U S A. 1985 Jul;82(14):4717–4721. [PubMed]
  • King SM, Otter T, Witman GB. Purification and characterization of Chlamydomonas flagellar dyneins. Methods Enzymol. 1986;134:291–306. [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]
  • King SM, Wilkerson CG, Witman GB. 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]
  • Kunkel TA, Roberts JD, Zakour RA. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. [PubMed]
  • Lewis SA, Wang DH, Cowan NJ. Microtubule-associated protein MAP2 shares a microtubule binding motif with tau protein. Science. 1988 Nov 11;242(4880):936–939. [PubMed]
  • Mitchell DR, Brown KS. Sequence analysis of the Chlamydomonas alpha and beta dynein heavy chain genes. J Cell Sci. 1994 Mar;107(Pt 3):635–644. [PubMed]
  • Mitchell DR, 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. [PMC free article] [PubMed]
  • Mitchell DR, Rosenbaum JL. Protein-protein interactions in the 18S ATPase of Chlamydomonas outer dynein arms. Cell Motil Cytoskeleton. 1986;6(5):510–520. [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]
  • Neer EJ, Schmidt CJ, Nambudripad R, Smith TF. The ancient regulatory-protein family of WD-repeat proteins. Nature. 1994 Sep 22;371(6495):297–300. [PubMed]
  • Ogawa K, Kamiya R, Wilkerson CG, Witman GB. Interspecies conservation of outer arm dynein intermediate chain sequences defines two intermediate chain subclasses. Mol Biol Cell. 1995 Jun;6(6):685–696. [PMC free article] [PubMed]
  • Paschal BM, Mikami A, Pfister KK, Vallee RB. Homology of the 74-kD cytoplasmic dynein subunit with a flagellar dynein polypeptide suggests an intracellular targeting function. J Cell Biol. 1992 Sep;118(5):1133–1143. [PMC free article] [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, Fay RB, Witman GB. Purification and polypeptide composition of dynein ATPases from Chlamydomonas flagella. Cell Motil. 1982;2(6):525–547. [PubMed]
  • Piperno G, Luck DJ. Axonemal adenosine triphosphatases from flagella of Chlamydomonas reinhardtii. Purification of two dyneins. J Biol Chem. 1979 Apr 25;254(8):3084–3090. [PubMed]
  • Rost B, Sander C. Prediction of protein secondary structure at better than 70% accuracy. J Mol Biol. 1993 Jul 20;232(2):584–599. [PubMed]
  • Sakakibara H, Takada S, King SM, Witman GB, Kamiya R. A Chlamydomonas outer arm dynein mutant with a truncated beta heavy chain. J Cell Biol. 1993 Aug;122(3):653–661. [PMC free article] [PubMed]
  • Sale WS, Goodenough UW, Heuser JE. The substructure of isolated and in situ outer dynein arms of sea urchin sperm flagella. J Cell Biol. 1985 Oct;101(4):1400–1412. [PMC free article] [PubMed]
  • Schneider C, Newman RA, Sutherland DR, Asser U, Greaves MF. A one-step purification of membrane proteins using a high efficiency immunomatrix. J Biol Chem. 1982 Sep 25;257(18):10766–10769. [PubMed]
  • Schneider A, Hemphill A, Wyler T, Seebeck T. Large microtubule-associated protein of T. brucei has tandemly repeated, near-identical sequences. Science. 1988 Jul 22;241(4864):459–462. [PubMed]
  • Takada S, Kamiya R. Functional reconstitution of Chlamydomonas outer dynein arms from alpha-beta and gamma subunits: requirement of a third factor. J Cell Biol. 1994 Aug;126(3):737–745. [PMC free article] [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]
  • 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]
  • Wilkerson CG, King SM, Witman GB. Molecular analysis of the gamma heavy chain of Chlamydomonas flagellar outer-arm dynein. J Cell Sci. 1994 Mar;107(Pt 3):497–506. [PubMed]
  • Wilkerson CG, King SM, Koutoulis A, Pazour GJ, Witman GB. The 78,000 M(r) intermediate chain of Chlamydomonas outer arm dynein isa WD-repeat protein required for arm assembly. J Cell Biol. 1995 Apr;129(1):169–178. [PMC free article] [PubMed]
  • Witman GB. Isolation of Chlamydomonas flagella and flagellar axonemes. Methods Enzymol. 1986;134:280–290. [PubMed]

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