Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1992 Sep 1;118(5):1133–1143. doi: 10.1083/jcb.118.5.1133

Homology of the 74-kD cytoplasmic dynein subunit with a flagellar dynein polypeptide suggests an intracellular targeting function

PMCID: PMC2289596  PMID: 1387402

Abstract

In previous work we found cytoplasmic dynein to be a complex of two catalytic heavy chains and at least seven co-purifying polypeptides of unknown function. The most prominent of these is a 74-kD electrophoretic species which can be resolved as two to three bands by SDS-PAGE. We have now selected a series of overlapping rat brain cDNAs encoding the 74-kD species. The deduced sequence of a full-length cDNA predicts a 72,753 D polypeptide which includes the amino acid sequences of nine peptides determined by NH2-terminal microsequencing. PCR performed on first strand rat brain cDNA together with the sequence of a partially matching tryptic peptide indicated the existence of at least three isoforms of the 74-kD cytoplasmic dynein subunit. Comparison with known sequences revealed that the carboxyl-terminal half of the polypeptide is 26.4% identical and 47.7% similar to the product of the Chlamydomonas ODA6 gene, a 70-kD intermediate chain of flagellar outer arm dynein. Immunoblot analysis with a monoclonal antibody to the 74-kD species indicated a widespread tissue distribution, as expected for a cytoplasmic dynein subunit. Nonetheless, the antibody recognized a 67-kD species in ram sperm flagella and pig tracheal cilia, supporting the existence of distinct but related cytoplasmic and axonemal polypeptides in mammals. In view of evidence for a role for the ODA6 gene product in anchoring flagellar dynein to the A subfiber microtubule in the axoneme, we predict an analogous role for the 74-kD polypeptide, perhaps in mediating the interaction of cytoplasmic dynein with membranous organelles and kinetochores.

Full Text

The Full Text of this article is available as a PDF (1.6 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Brendel V., Karlin S. Association of charge clusters with functional domains of cellular transcription factors. Proc Natl Acad Sci U S A. 1989 Aug;86(15):5698–5702. doi: 10.1073/pnas.86.15.5698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Calvin H. I. Isolation of subfractionation of mammalian sperm heads and tails. Methods Cell Biol. 1976;13:85–104. doi: 10.1016/s0091-679x(08)61798-7. [DOI] [PubMed] [Google Scholar]
  3. Collins C. A., Vallee R. B. Preparation of microtubules from rat liver and testis: cytoplasmic dynein is a major microtubule associated protein. Cell Motil Cytoskeleton. 1989;14(4):491–500. doi: 10.1002/cm.970140407. [DOI] [PubMed] [Google Scholar]
  4. Emini E. A., Hughes J. V., Perlow D. S., Boger J. Induction of hepatitis A virus-neutralizing antibody by a virus-specific synthetic peptide. J Virol. 1985 Sep;55(3):836–839. doi: 10.1128/jvi.55.3.836-839.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Gill S. R., Schroer T. A., Szilak I., Steuer E. R., Sheetz M. P., Cleveland D. W. Dynactin, a conserved, ubiquitously expressed component of an activator of vesicle motility mediated by cytoplasmic dynein. J Cell Biol. 1991 Dec;115(6):1639–1650. doi: 10.1083/jcb.115.6.1639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Hastie A. T. Purification and characterization of dynein from pig tracheal cilia. Methods Enzymol. 1991;196:223–234. doi: 10.1016/0076-6879(91)96021-i. [DOI] [PubMed] [Google Scholar]
  8. Holzbaur E. L., Hammarback J. A., Paschal B. M., Kravit N. G., Pfister K. K., Vallee R. B. Homology of a 150K cytoplasmic dynein-associated polypeptide with the Drosophila gene Glued. Nature. 1991 Jun 13;351(6327):579–583. doi: 10.1038/351579a0. [DOI] [PubMed] [Google Scholar]
  9. Hyman A. A., Mitchison T. J. Two different microtubule-based motor activities with opposite polarities in kinetochores. Nature. 1991 May 16;351(6323):206–211. doi: 10.1038/351206a0. [DOI] [PubMed] [Google Scholar]
  10. Johnson K. A. Pathway of the microtubule-dynein ATPase and the structure of dynein: a comparison with actomyosin. Annu Rev Biophys Biophys Chem. 1985;14:161–188. doi: 10.1146/annurev.bb.14.060185.001113. [DOI] [PubMed] [Google Scholar]
  11. Johnson K. A., Wall J. S. Structure and molecular weight of the dynein ATPase. J Cell Biol. 1983 Mar;96(3):669–678. doi: 10.1083/jcb.96.3.669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. King S. M., Gatti J. L., Moss A. G., Witman G. B. Outer-arm dynein from trout spermatozoa: substructural organization. Cell Motil Cytoskeleton. 1990;16(4):266–278. doi: 10.1002/cm.970160406. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. Kozak M. An analysis of vertebrate mRNA sequences: intimations of translational control. J Cell Biol. 1991 Nov;115(4):887–903. doi: 10.1083/jcb.115.4.887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lacey M. L., Haimo L. T. Cytoplasmic dynein is a vesicle protein. J Biol Chem. 1992 Mar 5;267(7):4793–4798. [PubMed] [Google Scholar]
  18. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  19. Lathe R. Synthetic oligonucleotide probes deduced from amino acid sequence data. Theoretical and practical considerations. J Mol Biol. 1985 May 5;183(1):1–12. doi: 10.1016/0022-2836(85)90276-1. [DOI] [PubMed] [Google Scholar]
  20. Lin S. X., Collins C. A. Immunolocalization of cytoplasmic dynein to lysosomes in cultured cells. J Cell Sci. 1992 Jan;101(Pt 1):125–137. doi: 10.1242/jcs.101.1.125. [DOI] [PubMed] [Google Scholar]
  21. Lipman D. J., Pearson W. R. Rapid and sensitive protein similarity searches. Science. 1985 Mar 22;227(4693):1435–1441. doi: 10.1126/science.2983426. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Matteoni R., Kreis T. E. Translocation and clustering of endosomes and lysosomes depends on microtubules. J Cell Biol. 1987 Sep;105(3):1253–1265. doi: 10.1083/jcb.105.3.1253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. Neely M. D., Boekelheide K. Sertoli cell processes have axoplasmic features: an ordered microtubule distribution and an abundant high molecular weight microtubule-associated protein (cytoplasmic dynein). J Cell Biol. 1988 Nov;107(5):1767–1776. doi: 10.1083/jcb.107.5.1767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Nudel U., Zakut R., Shani M., Neuman S., Levy Z., Yaffe D. The nucleotide sequence of the rat cytoplasmic beta-actin gene. Nucleic Acids Res. 1983 Mar 25;11(6):1759–1771. doi: 10.1093/nar/11.6.1759. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Oakley B. R., Kirsch D. R., Morris N. R. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem. 1980 Jul 1;105(2):361–363. doi: 10.1016/0003-2697(80)90470-4. [DOI] [PubMed] [Google Scholar]
  29. Paschal B. M., Shpetner H. S., Vallee R. B. MAP 1C is a microtubule-activated ATPase which translocates microtubules in vitro and has dynein-like properties. J Cell Biol. 1987 Sep;105(3):1273–1282. doi: 10.1083/jcb.105.3.1273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Paschal B. M., Shpetner H. S., Vallee R. B. Purification of brain cytoplasmic dynein and characterization of its in vitro properties. Methods Enzymol. 1991;196:181–191. doi: 10.1016/0076-6879(91)96018-m. [DOI] [PubMed] [Google Scholar]
  31. Paschal B. M., Vallee R. B. Retrograde transport by the microtubule-associated protein MAP 1C. Nature. 1987 Nov 12;330(6144):181–183. doi: 10.1038/330181a0. [DOI] [PubMed] [Google Scholar]
  32. Pfarr C. M., Coue M., Grissom P. M., Hays T. S., Porter M. E., McIntosh J. R. Cytoplasmic dynein is localized to kinetochores during mitosis. Nature. 1990 May 17;345(6272):263–265. doi: 10.1038/345263a0. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Rieder C. L., Alexander S. P. Kinetochores are transported poleward along a single astral microtubule during chromosome attachment to the spindle in newt lung cells. J Cell Biol. 1990 Jan;110(1):81–95. doi: 10.1083/jcb.110.1.81. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Robbins J., Dilworth S. M., Laskey R. A., Dingwall C. Two interdependent basic domains in nucleoplasmin nuclear targeting sequence: identification of a class of bipartite nuclear targeting sequence. Cell. 1991 Feb 8;64(3):615–623. doi: 10.1016/0092-8674(91)90245-t. [DOI] [PubMed] [Google Scholar]
  36. Rogalski A. A., Singer S. J. Associations of elements of the Golgi apparatus with microtubules. J Cell Biol. 1984 Sep;99(3):1092–1100. doi: 10.1083/jcb.99.3.1092. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Schroer T. A., Sheetz M. P. Two activators of microtubule-based vesicle transport. J Cell Biol. 1991 Dec;115(5):1309–1318. doi: 10.1083/jcb.115.5.1309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Shpetner H. S., Paschal B. M., Vallee R. B. Characterization of the microtubule-activated ATPase of brain cytoplasmic dynein (MAP 1C). J Cell Biol. 1988 Sep;107(3):1001–1009. doi: 10.1083/jcb.107.3.1001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Steuer E. R., Wordeman L., Schroer T. A., Sheetz M. P. Localization of cytoplasmic dynein to mitotic spindles and kinetochores. Nature. 1990 May 17;345(6272):266–268. doi: 10.1038/345266a0. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES