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. 1993 Aug 1;122(3):565–578. doi: 10.1083/jcb.122.3.565

Effects of mutant rat dynamin on endocytosis

PMCID: PMC2119668  PMID: 8335685

Abstract

Dynamin is a 100-kD microtubule-activated GTPase. Recent evidence has revealed a high degree of sequence homology with the product of the Drosophila gene shibire, mutations in which block the recycling of synaptic vesicles and, more generally, the formation of coated and non- coated vesicles at the plasma membrane. We have now transfected cultured mammalian COS-7 cells with both wild-type and mutant dynamin cDNAs. Point mutations in the GTP-binding consensus sequence elements of dynamin equivalent to dominant negative mutations in ras, and an NH2- terminal deletion of the entire GTP-binding domain of dynamin, block transferrin uptake and alter the distribution of clathrin heavy chain and alpha-, but not gamma-, adaptin. COOH-terminal deletions reverse these effects, identifying this portion of dynamin as a site of interaction with other components of the endocytic pathway. Over- expression of neither wild-type nor mutant forms of dynamin affected the distribution of microtubules. These results demonstrate a specific role for dynamin and for GTP in the initial stages of receptor-mediated endocytosis.

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Selected References

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  1. Ahle S., Mann A., Eichelsbacher U., Ungewickell E. Structural relationships between clathrin assembly proteins from the Golgi and the plasma membrane. EMBO J. 1988 Apr;7(4):919–929. doi: 10.1002/j.1460-2075.1988.tb02897.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bamburg J. R., Shooter E. M., Wilson L. Developmental changes in microtubule protein of chick brain. Biochemistry. 1973 Apr 10;12(8):1476–1482. doi: 10.1021/bi00732a002. [DOI] [PubMed] [Google Scholar]
  3. Bourne H. R., Sanders D. A., McCormick F. The GTPase superfamily: conserved structure and molecular mechanism. Nature. 1991 Jan 10;349(6305):117–127. doi: 10.1038/349117a0. [DOI] [PubMed] [Google Scholar]
  4. Brodsky F. M. Living with clathrin: its role in intracellular membrane traffic. Science. 1988 Dec 9;242(4884):1396–1402. doi: 10.1126/science.2904698. [DOI] [PubMed] [Google Scholar]
  5. Carter L. L., Redelmeier T. E., Woollenweber L. A., Schmid S. L. Multiple GTP-binding proteins participate in clathrin-coated vesicle-mediated endocytosis. J Cell Biol. 1993 Jan;120(1):37–45. doi: 10.1083/jcb.120.1.37. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chen M. S., Burgess C. C., Vallee R. B., Wadsworth S. C. Developmental stage- and tissue-specific expression of shibire, a Drosophila gene involved in endocytosis. J Cell Sci. 1992 Nov;103(Pt 3):619–628. doi: 10.1242/jcs.103.3.619. [DOI] [PubMed] [Google Scholar]
  7. Chen M. S., Obar R. A., Schroeder C. C., Austin T. W., Poodry C. A., Wadsworth S. C., Vallee R. B. Multiple forms of dynamin are encoded by shibire, a Drosophila gene involved in endocytosis. Nature. 1991 Jun 13;351(6327):583–586. doi: 10.1038/351583a0. [DOI] [PubMed] [Google Scholar]
  8. Doxsey S. J., Brodsky F. M., Blank G. S., Helenius A. Inhibition of endocytosis by anti-clathrin antibodies. Cell. 1987 Jul 31;50(3):453–463. doi: 10.1016/0092-8674(87)90499-5. [DOI] [PubMed] [Google Scholar]
  9. Draper R. K., Goda Y., Brodsky F. M., Pfeffer S. R. Antibodies to clathrin inhibit endocytosis but not recycling to the trans Golgi network in vitro. Science. 1990 Jun 22;248(4962):1539–1541. doi: 10.1126/science.2163108. [DOI] [PubMed] [Google Scholar]
  10. Farnsworth C. L., Feig L. A. Dominant inhibitory mutations in the Mg(2+)-binding site of RasH prevent its activation by GTP. Mol Cell Biol. 1991 Oct;11(10):4822–4829. doi: 10.1128/mcb.11.10.4822. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Feig L. A., Cooper G. M. Inhibition of NIH 3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP. Mol Cell Biol. 1988 Aug;8(8):3235–3243. doi: 10.1128/mcb.8.8.3235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Feig L. A., Pan B. T., Roberts T. M., Cooper G. M. Isolation of ras GTP-binding mutants using an in situ colony-binding assay. Proc Natl Acad Sci U S A. 1986 Jul;83(13):4607–4611. doi: 10.1073/pnas.83.13.4607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Flucher B. E., Terasaki M., Chin H. M., Beeler T. J., Daniels M. P. Biogenesis of transverse tubules in skeletal muscle in vitro. Dev Biol. 1991 May;145(1):77–90. doi: 10.1016/0012-1606(91)90214-n. [DOI] [PubMed] [Google Scholar]
  14. Grigliatti T. A., Hall L., Rosenbluth R., Suzuki D. T. Temperature-sensitive mutations in Drosophila melanogaster. XIV. A selection of immobile adults. Mol Gen Genet. 1973 Jan 24;120(2):107–114. doi: 10.1007/BF00267238. [DOI] [PubMed] [Google Scholar]
  15. Honig M. G., Hume R. I. Fluorescent carbocyanine dyes allow living neurons of identified origin to be studied in long-term cultures. J Cell Biol. 1986 Jul;103(1):171–187. doi: 10.1083/jcb.103.1.171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Horisberger M. A., Gunst M. C. Interferon-induced proteins: identification of Mx proteins in various mammalian species. Virology. 1991 Jan;180(1):185–190. doi: 10.1016/0042-6822(91)90022-4. [DOI] [PubMed] [Google Scholar]
  17. Horisberger M. A. Interferon-induced human protein MxA is a GTPase which binds transiently to cellular proteins. J Virol. 1992 Aug;66(8):4705–4709. doi: 10.1128/jvi.66.8.4705-4709.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Horisberger M. A., McMaster G. K., Zeller H., Wathelet M. G., Dellis J., Content J. Cloning and sequence analyses of cDNAs for interferon- and virus-induced human Mx proteins reveal that they contain putative guanine nucleotide-binding sites: functional study of the corresponding gene promoter. J Virol. 1990 Mar;64(3):1171–1181. doi: 10.1128/jvi.64.3.1171-1181.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Horisberger M. A., Staeheli P., Haller O. Interferon induces a unique protein in mouse cells bearing a gene for resistance to influenza virus. Proc Natl Acad Sci U S A. 1983 Apr;80(7):1910–1914. doi: 10.1073/pnas.80.7.1910. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jones B. A., Fangman W. L. Mitochondrial DNA maintenance in yeast requires a protein containing a region related to the GTP-binding domain of dynamin. Genes Dev. 1992 Mar;6(3):380–389. doi: 10.1101/gad.6.3.380. [DOI] [PubMed] [Google Scholar]
  21. Kahn R. A., Goddard C., Newkirk M. Chemical and immunological characterization of the 21-kDa ADP-ribosylation factor of adenylate cyclase. J Biol Chem. 1988 Jun 15;263(17):8282–8287. [PubMed] [Google Scholar]
  22. Kahn R. A., Kern F. G., Clark J., Gelmann E. P., Rulka C. Human ADP-ribosylation factors. A functionally conserved family of GTP-binding proteins. J Biol Chem. 1991 Feb 5;266(4):2606–2614. [PubMed] [Google Scholar]
  23. Kahn R. A., Randazzo P., Serafini T., Weiss O., Rulka C., Clark J., Amherdt M., Roller P., Orci L., Rothman J. E. The amino terminus of ADP-ribosylation factor (ARF) is a critical determinant of ARF activities and is a potent and specific inhibitor of protein transport. J Biol Chem. 1992 Jun 25;267(18):13039–13046. [PubMed] [Google Scholar]
  24. Keen J. H. Clathrin and associated assembly and disassembly proteins. Annu Rev Biochem. 1990;59:415–438. doi: 10.1146/annurev.bi.59.070190.002215. [DOI] [PubMed] [Google Scholar]
  25. Kessell I., Holst B. D., Roth T. F. Membranous intermediates in endocytosis are labile, as shown in a temperature-sensitive mutant. Proc Natl Acad Sci U S A. 1989 Jul;86(13):4968–4972. doi: 10.1073/pnas.86.13.4968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kim Y. T., Wu C. F. Allelic interactions at the shibire locus of Drosophila: effects on behavior. J Neurogenet. 1990 Nov;7(1):1–14. doi: 10.3109/01677069009084149. [DOI] [PubMed] [Google Scholar]
  27. Koenig J. H., Ikeda K. Disappearance and reformation of synaptic vesicle membrane upon transmitter release observed under reversible blockage of membrane retrieval. J Neurosci. 1989 Nov;9(11):3844–3860. doi: 10.1523/JNEUROSCI.09-11-03844.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Koenig J. H., Saito K., Ikeda K. Reversible control of synaptic transmission in a single gene mutant of Drosophila melanogaster. J Cell Biol. 1983 Jun;96(6):1517–1522. doi: 10.1083/jcb.96.6.1517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kosaka T., Ikeda K. Possible temperature-dependent blockage of synaptic vesicle recycling induced by a single gene mutation in Drosophila. J Neurobiol. 1983 May;14(3):207–225. doi: 10.1002/neu.480140305. [DOI] [PubMed] [Google Scholar]
  30. Kosaka T., Ikeda K. Reversible blockage of membrane retrieval and endocytosis in the garland cell of the temperature-sensitive mutant of Drosophila melanogaster, shibirets1. J Cell Biol. 1983 Aug;97(2):499–507. doi: 10.1083/jcb.97.2.499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Lin H. C., Moore M. S., Sanan D. A., Anderson R. G. Reconstitution of clathrin-coated pit budding from plasma membranes. J Cell Biol. 1991 Sep;114(5):881–891. doi: 10.1083/jcb.114.5.881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. Lippincott-Schwartz J., Donaldson J. G., Schweizer A., Berger E. G., Hauri H. P., Yuan L. C., Klausner R. D. Microtubule-dependent retrograde transport of proteins into the ER in the presence of brefeldin A suggests an ER recycling pathway. Cell. 1990 Mar 9;60(5):821–836. doi: 10.1016/0092-8674(90)90096-w. [DOI] [PubMed] [Google Scholar]
  35. Lippincott-Schwartz J., Yuan L., Tipper C., Amherdt M., Orci L., Klausner R. D. Brefeldin A's effects on endosomes, lysosomes, and the TGN suggest a general mechanism for regulating organelle structure and membrane traffic. Cell. 1991 Nov 1;67(3):601–616. doi: 10.1016/0092-8674(91)90534-6. [DOI] [PubMed] [Google Scholar]
  36. Masur S. K., Kim Y. T., Wu C. F. Reversible inhibition of endocytosis in cultured neurons from the Drosophila temperature-sensitive mutant shibirets1. J Neurogenet. 1990 Apr;6(3):191–206. doi: 10.3109/01677069009107110. [DOI] [PubMed] [Google Scholar]
  37. Nakata T., Iwamoto A., Noda Y., Takemura R., Yoshikura H., Hirokawa N. Predominant and developmentally regulated expression of dynamin in neurons. Neuron. 1991 Sep;7(3):461–469. doi: 10.1016/0896-6273(91)90298-e. [DOI] [PubMed] [Google Scholar]
  38. Nakayama M., Nagata K., Kato A., Ishihama A. Interferon-inducible mouse Mx1 protein that confers resistance to influenza virus is GTPase. J Biol Chem. 1991 Nov 15;266(32):21404–21408. [PubMed] [Google Scholar]
  39. Obar R. A., Collins C. A., Hammarback J. A., Shpetner H. S., Vallee R. B. Molecular cloning of the microtubule-associated mechanochemical enzyme dynamin reveals homology with a new family of GTP-binding proteins. Nature. 1990 Sep 20;347(6290):256–261. doi: 10.1038/347256a0. [DOI] [PubMed] [Google Scholar]
  40. Pai E. F., Kabsch W., Krengel U., Holmes K. C., John J., Wittinghofer A. Structure of the guanine-nucleotide-binding domain of the Ha-ras oncogene product p21 in the triphosphate conformation. Nature. 1989 Sep 21;341(6239):209–214. doi: 10.1038/341209a0. [DOI] [PubMed] [Google Scholar]
  41. Pai E. F., Krengel U., Petsko G. A., Goody R. S., Kabsch W., Wittinghofer A. Refined crystal structure of the triphosphate conformation of H-ras p21 at 1.35 A resolution: implications for the mechanism of GTP hydrolysis. EMBO J. 1990 Aug;9(8):2351–2359. doi: 10.1002/j.1460-2075.1990.tb07409.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Pearse B. M., Robinson M. S. Clathrin, adaptors, and sorting. Annu Rev Cell Biol. 1990;6:151–171. doi: 10.1146/annurev.cb.06.110190.001055. [DOI] [PubMed] [Google Scholar]
  43. Pfister K. K., Wagner M. C., Stenoien D. L., Brady S. T., Bloom G. S. Monoclonal antibodies to kinesin heavy and light chains stain vesicle-like structures, but not microtubules, in cultured cells. J Cell Biol. 1989 Apr;108(4):1453–1463. doi: 10.1083/jcb.108.4.1453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Poodry C. A., Edgar L. Reversible alteration in the neuromuscular junctions of Drosophila melanogaster bearing a temperature-sensitive mutation, shibire. J Cell Biol. 1979 Jun;81(3):520–527. doi: 10.1083/jcb.81.3.520. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Robinson M. S., Kreis T. E. Recruitment of coat proteins onto Golgi membranes in intact and permeabilized cells: effects of brefeldin A and G protein activators. Cell. 1992 Apr 3;69(1):129–138. doi: 10.1016/0092-8674(92)90124-u. [DOI] [PubMed] [Google Scholar]
  46. Robinson M. S., Pearse B. M. Immunofluorescent localization of 100K coated vesicle proteins. J Cell Biol. 1986 Jan;102(1):48–54. doi: 10.1083/jcb.102.1.48. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Rothberg K. G., Heuser J. E., Donzell W. C., Ying Y. S., Glenney J. R., Anderson R. G. Caveolin, a protein component of caveolae membrane coats. Cell. 1992 Feb 21;68(4):673–682. doi: 10.1016/0092-8674(92)90143-z. [DOI] [PubMed] [Google Scholar]
  48. Rothberg K. G., Ying Y. S., Kolhouse J. F., Kamen B. A., Anderson R. G. The glycophospholipid-linked folate receptor internalizes folate without entering the clathrin-coated pit endocytic pathway. J Cell Biol. 1990 Mar;110(3):637–649. doi: 10.1083/jcb.110.3.637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Rothman J. H., Raymond C. K., Gilbert T., O'Hara P. J., Stevens T. H. A putative GTP binding protein homologous to interferon-inducible Mx proteins performs an essential function in yeast protein sorting. Cell. 1990 Jun 15;61(6):1063–1074. doi: 10.1016/0092-8674(90)90070-u. [DOI] [PubMed] [Google Scholar]
  50. Sandvig K., van Deurs B. Selective modulation of the endocytic uptake of ricin and fluid phase markers without alteration in transferrin endocytosis. J Biol Chem. 1990 Apr 15;265(11):6382–6388. [PubMed] [Google Scholar]
  51. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Scaife R., Margolis R. L. Biochemical and immunochemical analysis of rat brain dynamin interaction with microtubules and organelles in vivo and in vitro. J Cell Biol. 1990 Dec;111(6 Pt 2):3023–3033. doi: 10.1083/jcb.111.6.3023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Schmid S. L., Smythe E. Stage-specific assays for coated pit formation and coated vesicle budding in vitro. J Cell Biol. 1991 Sep;114(5):869–880. doi: 10.1083/jcb.114.5.869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Shpetner H. S., Vallee R. B. Dynamin is a GTPase stimulated to high levels of activity by microtubules. Nature. 1992 Feb 20;355(6362):733–735. doi: 10.1038/355733a0. [DOI] [PubMed] [Google Scholar]
  55. Shpetner H. S., Vallee R. B. Identification of dynamin, a novel mechanochemical enzyme that mediates interactions between microtubules. Cell. 1989 Nov 3;59(3):421–432. doi: 10.1016/0092-8674(89)90027-5. [DOI] [PubMed] [Google Scholar]
  56. Sigal I. S., Gibbs J. B., D'Alonzo J. S., Temeles G. L., Wolanski B. S., Socher S. H., Scolnick E. M. Mutant ras-encoded proteins with altered nucleotide binding exert dominant biological effects. Proc Natl Acad Sci U S A. 1986 Feb;83(4):952–956. doi: 10.1073/pnas.83.4.952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Staeheli P., Haller O., Boll W., Lindenmann J., Weissmann C. Mx protein: constitutive expression in 3T3 cells transformed with cloned Mx cDNA confers selective resistance to influenza virus. Cell. 1986 Jan 17;44(1):147–158. doi: 10.1016/0092-8674(86)90493-9. [DOI] [PubMed] [Google Scholar]
  58. 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]
  59. Vallee R. B., Collins C. A. Purification of microtubules and microtubule-associated proteins from sea urchin eggs and cultured mammalian cells using taxol, and use of exogenous taxol-stabilized brain microtubules for purifying microtubule-associated proteins. Methods Enzymol. 1986;134:116–127. doi: 10.1016/0076-6879(86)34080-1. [DOI] [PubMed] [Google Scholar]
  60. Walworth N. C., Brennwald P., Kabcenell A. K., Garrett M., Novick P. Hydrolysis of GTP by Sec4 protein plays an important role in vesicular transport and is stimulated by a GTPase-activating protein in Saccharomyces cerevisiae. Mol Cell Biol. 1992 May;12(5):2017–2028. doi: 10.1128/mcb.12.5.2017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Yeh E., Driscoll R., Coltrera M., Olins A., Bloom K. A dynamin-like protein encoded by the yeast sporulation gene SPO15. Nature. 1991 Feb 21;349(6311):713–715. doi: 10.1038/349713a0. [DOI] [PubMed] [Google Scholar]
  62. van der Bliek A. M., Meyerowitz E. M. Dynamin-like protein encoded by the Drosophila shibire gene associated with vesicular traffic. Nature. 1991 May 30;351(6325):411–414. doi: 10.1038/351411a0. [DOI] [PubMed] [Google Scholar]

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