Search tips
Search criteria 


Logo of jcellbiolHomeThe Rockefeller University PressEditorsContactInstructions for AuthorsThis issue
J Cell Biol. 1994 November 2; 127(4): 915–934.
PMCID: PMC2200053

Induction of mutant dynamin specifically blocks endocytic coated vesicle formation


Dynamin is the mammalian homologue to the Drosophila shibire gene product. Mutations in this 100-kD GTPase cause a pleiotropic defect in endocytosis. To further investigate its role, we generated stable HeLa cell lines expressing either wild-type dynamin or a mutant defective in GTP binding and hydrolysis driven by a tightly controlled, tetracycline- inducible promoter. Overexpression of wild-type dynamin had no effect. In contrast, coated pits failed to become constricted and coated vesicles failed to bud in cells overexpressing mutant dynamin so that endocytosis via both transferrin (Tfn) and EGF receptors was potently inhibited. Coated pit assembly, invagination, and the recruitment of receptors into coated pits were unaffected. Other vesicular transport pathways, including Tfn receptor recycling, Tfn receptor biosynthesis, and cathepsin D transport to lysosomes via Golgi-derived coated vesicles, were unaffected. Bulk fluid-phase uptake also continued at the same initial rates as wild type. EM immunolocalization showed that membrane-bound dynamin was specifically associated with clathrin-coated pits on the plasma membrane. Dynamin was also associated with isolated coated vesicles, suggesting that it plays a role in vesicle budding. Like the Drosophila shibire mutant, HeLa cells overexpressing mutant dynamin accumulated long tubules, many of which remained connected to the plasma membrane. We conclude that dynamin is specifically required for endocytic coated vesicle formation, and that its GTP binding and hydrolysis activities are required to form constricted coated pits and, subsequently, for coated vesicle budding.

Full Text

The Full Text of this article is available as a PDF (6.6M).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • 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. [PubMed]
  • Altankov G, Grinnell F. Depletion of intracellular potassium disrupts coated pits and reversibly inhibits cell polarization during fibroblast spreading. J Cell Biol. 1993 Mar;120(6):1449–1459. [PMC free article] [PubMed]
  • Balch WE, McCaffery JM, Plutner H, Farquhar MG. Vesicular stomatitis virus glycoprotein is sorted and concentrated during export from the endoplasmic reticulum. Cell. 1994 Mar 11;76(5):841–852. [PubMed]
  • Benfenati F, Bähler M, Jahn R, Greengard P. Interactions of synapsin I with small synaptic vesicles: distinct sites in synapsin I bind to vesicle phospholipids and vesicle proteins. J Cell Biol. 1989 May;108(5):1863–1872. [PMC free article] [PubMed]
  • Benfenati F, Valtorta F, Rossi MC, Onofri F, Sihra T, Greengard P. Interactions of synapsin I with phospholipids: possible role in synaptic vesicle clustering and in the maintenance of bilayer structures. J Cell Biol. 1993 Dec;123(6 Pt 2):1845–1855. [PMC free article] [PubMed]
  • Besterman JM, Airhart JA, Woodworth RC, Low RB. Exocytosis of pinocytosed fluid in cultured cells: kinetic evidence for rapid turnover and compartmentation. J Cell Biol. 1981 Dec;91(3 Pt 1):716–727. [PMC free article] [PubMed]
  • Burnette WN. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. [PubMed]
  • Buzin CH, Dewhurst SA, Seecof RL. Temperature sensitivity of muscle and neuron differentiation in embryonic cell cultures from the Drosophila mutant, shibire. Dev Biol. 1978 Oct;66(2):442–456. [PubMed]
  • Carter LL, Redelmeier TE, Woollenweber LA, Schmid SL. Multiple GTP-binding proteins participate in clathrin-coated vesicle-mediated endocytosis. J Cell Biol. 1993 Jan;120(1):37–45. [PMC free article] [PubMed]
  • Chen MS, Obar RA, Schroeder CC, Austin TW, Poodry CA, Wadsworth SC, Vallee RB. Multiple forms of dynamin are encoded by shibire, a Drosophila gene involved in endocytosis. Nature. 1991 Jun 13;351(6327):583–586. [PubMed]
  • Ciechanover A, Schwartz AL, Dautry-Varsat A, Lodish HF. Kinetics of internalization and recycling of transferrin and the transferrin receptor in a human hepatoma cell line. Effect of lysosomotropic agents. J Biol Chem. 1983 Aug 25;258(16):9681–9689. [PubMed]
  • Collins CA. Dynamin: a novel microtubule-associated GTPase. Trends Cell Biol. 1991 Aug;1(2-3):57–60. [PubMed]
  • Cook TA, Urrutia R, McNiven MA. Identification of dynamin 2, an isoform ubiquitously expressed in rat tissues. Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):644–648. [PubMed]
  • de la Luna S, Soria I, Pulido D, Ortín J, Jiménez A. Efficient transformation of mammalian cells with constructs containing a puromycin-resistance marker. Gene. 1988;62(1):121–126. [PubMed]
  • Gilmore R. The protein translocation apparatus of the rough endoplasmic reticulum, its associated proteins, and the mechanism of translocation. Curr Opin Cell Biol. 1991 Aug;3(4):580–584. [PubMed]
  • Gossen M, Bujard H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5547–5551. [PubMed]
  • Gout I, Dhand R, Hiles ID, Fry MJ, Panayotou G, Das P, Truong O, Totty NF, Hsuan J, Booker GW, et al. The GTPase dynamin binds to and is activated by a subset of SH3 domains. Cell. 1993 Oct 8;75(1):25–36. [PubMed]
  • Griffiths G, Back R, Marsh M. A quantitative analysis of the endocytic pathway in baby hamster kidney cells. J Cell Biol. 1989 Dec;109(6 Pt 1):2703–2720. [PMC free article] [PubMed]
  • Grigliatti TA, Hall L, Rosenbluth R, Suzuki DT. Temperature-sensitive mutations in Drosophila melanogaster. XIV. A selection of immobile adults. Mol Gen Genet. 1973 Jan 24;120(2):107–114. [PubMed]
  • Herskovits JS, Burgess CC, Obar RA, Vallee RB. Effects of mutant rat dynamin on endocytosis. J Cell Biol. 1993 Aug;122(3):565–578. [PMC free article] [PubMed]
  • Herskovits JS, Shpetner HS, Burgess CC, Vallee RB. Microtubules and Src homology 3 domains stimulate the dynamin GTPase via its C-terminal domain. Proc Natl Acad Sci U S A. 1993 Dec 15;90(24):11468–11472. [PubMed]
  • Heuser J. Three-dimensional visualization of coated vesicle formation in fibroblasts. J Cell Biol. 1980 Mar;84(3):560–583. [PMC free article] [PubMed]
  • Kessell I, Holst BD, Roth TF. 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. [PubMed]
  • Kim YT, Wu CF. Reversible blockage of neurite development and growth cone formation in neuronal cultures of a temperature-sensitive mutant of Drosophila. J Neurosci. 1987 Oct;7(10):3245–3255. [PubMed]
  • Koenig JH, 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. [PubMed]
  • Koenig JH, Ikeda K. Transformational process of the endosomal compartment in nephrocytes of Drosophila melanogaster. Cell Tissue Res. 1990 Nov;262(2):233–244. [PubMed]
  • Kornfeld S, Mellman I. The biogenesis of lysosomes. Annu Rev Cell Biol. 1989;5:483–525. [PubMed]
  • 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. [PubMed]
  • 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. [PMC free article] [PubMed]
  • Lamaze C, Baba T, Redelmeier TE, Schmid SL. Recruitment of epidermal growth factor and transferrin receptors into coated pits in vitro: differing biochemical requirements. Mol Biol Cell. 1993 Jul;4(7):715–727. [PMC free article] [PubMed]
  • Maeda K, Nakata T, Noda Y, Sato-Yoshitake R, Hirokawa N. Interaction of dynamin with microtubules: its structure and GTPase activity investigated by using highly purified dynamin. Mol Biol Cell. 1992 Oct;3(10):1181–1194. [PMC free article] [PubMed]
  • Mahaffey DT, Peeler JS, Brodsky FM, Anderson RG. Clathrin-coated pits contain an integral membrane protein that binds the AP-2 subunit with high affinity. J Biol Chem. 1990 Sep 25;265(27):16514–16520. [PubMed]
  • Marsh M, Schmid S, Kern H, Harms E, Male P, Mellman I, Helenius A. Rapid analytical and preparative isolation of functional endosomes by free flow electrophoresis. J Cell Biol. 1987 Apr;104(4):875–886. [PMC free article] [PubMed]
  • Moore MS, Mahaffey DT, Brodsky FM, Anderson RG. Assembly of clathrin-coated pits onto purified plasma membranes. Science. 1987 May 1;236(4801):558–563. [PubMed]
  • 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. [PubMed]
  • Nakata T, Takemura R, Hirokawa N. A novel member of the dynamin family of GTP-binding proteins is expressed specifically in the testis. J Cell Sci. 1993 May;105(Pt 1):1–5. [PubMed]
  • Narita K, Tsuruhara T, Koenig JH, Ikeda K. Membrane pinch-off and reinsertion observed in living cells of Drosophila. J Cell Physiol. 1989 Nov;141(2):383–391. [PubMed]
  • Nuoffer C, Balch WE. GTPases: multifunctional molecular switches regulating vesicular traffic. Annu Rev Biochem. 1994;63:949–990. [PubMed]
  • Obar RA, Collins CA, Hammarback JA, Shpetner HS, Vallee RB. 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. [PubMed]
  • Oka T, Nakano A. Inhibition of GTP hydrolysis by Sar1p causes accumulation of vesicles that are a functional intermediate of the ER-to-Golgi transport in yeast. J Cell Biol. 1994 Feb;124(4):425–434. [PMC free article] [PubMed]
  • Omary MB, Trowbridge IS. Biosynthesis of the human transferrin receptor in cultured cells. J Biol Chem. 1981 Dec 25;256(24):12888–12892. [PubMed]
  • Pai EF, Krengel U, Petsko GA, Goody RS, 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. [PubMed]
  • Pearse BM. Coated vesicles from pig brain: purification and biochemical characterization. J Mol Biol. 1975 Sep 5;97(1):93–98. [PubMed]
  • Poodry CA, 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. [PMC free article] [PubMed]
  • Pypaert M, Lucocq JM, Warren G. Coated pits in interphase and mitotic A431 cells. Eur J Cell Biol. 1987 Dec;45(1):23–29. [PubMed]
  • Resnitzky D, Gossen M, Bujard H, Reed SI. Acceleration of the G1/S phase transition by expression of cyclins D1 and E with an inducible system. Mol Cell Biol. 1994 Mar;14(3):1669–1679. [PMC free article] [PubMed]
  • Robinson MS. Adaptins. Trends Cell Biol. 1992 Oct;2(10):293–297. [PubMed]
  • Robinson PJ, Sontag JM, Liu JP, Fykse EM, Slaughter C, McMahon H, Südhof TC. Dynamin GTPase regulated by protein kinase C phosphorylation in nerve terminals. Nature. 1993 Sep 9;365(6442):163–166. [PubMed]
  • Sanan DA, Anderson RG. Simultaneous visualization of LDL receptor distribution and clathrin lattices on membranes torn from the upper surface of cultured cells. J Histochem Cytochem. 1991 Aug;39(8):1017–1024. [PubMed]
  • Scaife R, Margolis RL. 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. [PMC free article] [PubMed]
  • Schmid SL, Carter LL. ATP is required for receptor-mediated endocytosis in intact cells. J Cell Biol. 1990 Dec;111(6 Pt 1):2307–2318. [PMC free article] [PubMed]
  • Schmid SL, Smythe E. Stage-specific assays for coated pit formation and coated vesicle budding in vitro. J Cell Biol. 1991 Sep;114(5):869–880. [PMC free article] [PubMed]
  • Schmid SL. Coated-vesicle formation in vitro: conflicting results using different assays. Trends Cell Biol. 1993 May;3(5):145–148. [PubMed]
  • Seeger M, Payne GS. A role for clathrin in the sorting of vacuolar proteins in the Golgi complex of yeast. EMBO J. 1992 Aug;11(8):2811–2818. [PubMed]
  • Shpetner HS, Vallee RB. Identification of dynamin, a novel mechanochemical enzyme that mediates interactions between microtubules. Cell. 1989 Nov 3;59(3):421–432. [PubMed]
  • Shpetner HS, Vallee RB. Dynamin is a GTPase stimulated to high levels of activity by microtubules. Nature. 1992 Feb 20;355(6362):733–735. [PubMed]
  • Smythe E, Pypaert M, Lucocq J, Warren G. Formation of coated vesicles from coated pits in broken A431 cells. J Cell Biol. 1989 Mar;108(3):843–853. [PMC free article] [PubMed]
  • Smythe E, Carter LL, Schmid SL. Cytosol- and clathrin-dependent stimulation of endocytosis in vitro by purified adaptors. J Cell Biol. 1992 Dec;119(5):1163–1171. [PMC free article] [PubMed]
  • Smythe E, Redelmeier TE, Schmid SL. Receptor-mediated endocytosis in semiintact cells. Methods Enzymol. 1992;219:223–234. [PubMed]
  • Sontag JM, Fykse EM, Ushkaryov Y, Liu JP, Robinson PJ, Südhof TC. Differential expression and regulation of multiple dynamins. J Biol Chem. 1994 Feb 11;269(6):4547–4554. [PubMed]
  • Trowbridge IS, Collawn JF, Hopkins CR. Signal-dependent membrane protein trafficking in the endocytic pathway. Annu Rev Cell Biol. 1993;9:129–161. [PubMed]
  • Tsuruhara T, Koenig JH, Ikeda K. Synchronized endocytosis studied in the oocyte of a temperature-sensitive mutant of Drosophila melanogaster. Cell Tissue Res. 1990 Feb;259(2):199–207. [PubMed]
  • Tuma PL, Stachniak MC, Collins CA. Activation of dynamin GTPase by acidic phospholipids and endogenous rat brain vesicles. J Biol Chem. 1993 Aug 15;268(23):17240–17246. [PubMed]
  • Vallee RB. Dynamin: motor protein or regulatory GTPase. J Muscle Res Cell Motil. 1992 Oct;13(5):493–496. [PubMed]
  • van der Bliek AM, Meyerowitz EM. Dynamin-like protein encoded by the Drosophila shibire gene associated with vesicular traffic. Nature. 1991 May 30;351(6325):411–414. [PubMed]
  • van der Bliek AM, Redelmeier TE, Damke H, Tisdale EJ, Meyerowitz EM, Schmid SL. Mutations in human dynamin block an intermediate stage in coated vesicle formation. J Cell Biol. 1993 Aug;122(3):553–563. [PMC free article] [PubMed]
  • van Deurs B, Petersen OW, Olsnes S, Sandvig K. The ways of endocytosis. Int Rev Cytol. 1989;117:131–177. [PubMed]
  • Vater CA, Raymond CK, Ekena K, Howald-Stevenson I, Stevens TH. The VPS1 protein, a homolog of dynamin required for vacuolar protein sorting in Saccharomyces cerevisiae, is a GTPase with two functionally separable domains. J Cell Biol. 1992 Nov;119(4):773–786. [PMC free article] [PubMed]
  • Williamson MP. The structure and function of proline-rich regions in proteins. Biochem J. 1994 Jan 15;297(Pt 2):249–260. [PubMed]
  • Wilsbach K, Payne GS. Vps1p, a member of the dynamin GTPase family, is necessary for Golgi membrane protein retention in Saccharomyces cerevisiae. EMBO J. 1993 Aug;12(8):3049–3059. [PubMed]
  • Wilson IA, Niman HL, Houghten RA, Cherenson AR, Connolly ML, Lerner RA. The structure of an antigenic determinant in a protein. Cell. 1984 Jul;37(3):767–778. [PubMed]
  • Zerial M, Stenmark H. Rab GTPases in vesicular transport. Curr Opin Cell Biol. 1993 Aug;5(4):613–620. [PubMed]

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