How We Study Protein Transport

Part of the Molecular Biology Intelligence Unit book series (MBIU)


For the greater part of the last century, research in the field of protein transport was synonymous with microscopy. Before the end of the century, this view was dramatically changed by the emergence of innovative genetic, molecular and biochemical approaches that revolutionized and invigorated the field. Far from being displaced as an essential tool, microscopy techniques have also evolved. What was once largely a science of “dead cells” has been transformed into a window on the inner workings of living cells. The objective of this chapter is to provide an overview of the major approaches that are employed in the analysis of protein transport within the membrane trafficking system of eukaryotic cells. In particular, we discuss the identification of several of the common model cargo proteins for studying both secretory and endocytic membrane trafficking in both mammalian and yeast systems. We then discuss the development of both in vivo and in vitro techniques to study the transport of these model cargo proteins within cells, and explain some of the common principles involved in these assays. Finally, we discuss some of the recent advances in imaging techniques and technology that have driven the recent “renaissance” in the use of microscopic techniques in the investigation of membrane trafficking events in living cells.


Green Fluorescent Protein Fluorescence Resonance Energy Transfer Membrane Trafficking Nonpermissive Temperature Cargo Protein 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


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  1. 1.
    Balch WE, Wagner KR, Keller DS. Reconstitution of transport of vesicular stomatitis virus G protein from the endoplasmic reticulum to the Golgi complex using a cell-free system. J Cell Biol 1987; 104(3):749–60.PubMedCrossRefGoogle Scholar
  2. 2.
    Schwaninger R, Beckers CJ, Balch WE. Sequential transport of protein between the endoplasmic reticulum and successive Golgi compartments in semi-intact cells. J Biol Chem 1991; 266:13055–63.PubMedGoogle Scholar
  3. 3.
    Keller P, Simons K. Post-Golgi biosynthetic trafficking. J Cell Sci 1997; 110(Part 24):3001–9.PubMedGoogle Scholar
  4. 4.
    Yoshimori T, Keller P, Roth MG et al. Different biosynthetic transport routes to the plasma membrane in BHK and CHO cells. J Cell Biol 1996; 133(2):247–56.PubMedCrossRefGoogle Scholar
  5. 5.
    Gallione CJ, Rose JK. A single amino acid substitution in a hydrophobic domain causes temperature-sensitive cell-surface transport of a mutant viral glycoprotein. J Virol 1985; 54(2):374–82.PubMedGoogle Scholar
  6. 6.
    Bergman J. Using temperature sensitive mutants of VSV to study membrane protein biogenesis. Meth Cell Biol 1989; 32:85–110.CrossRefGoogle Scholar
  7. 7.
    Kuismanen E, Saraste J. Low temperature-induced transport blocks as tools to manipulate membrane traffic. Methods Cell Biol 1989; 32:257–74.PubMedCrossRefGoogle Scholar
  8. 8.
    Saraste J, Palade GE, Farquhar MG. Temperature-sensitive steps in the transport of secretory proteins through the Golgi complex in exocrine pancreatic cells. Proc Natl Acad Sci USA 1986; 83(17):6425–9.PubMedCrossRefGoogle Scholar
  9. 9.
    Rose JK, Bergmann JE. Expression from cloned cDNA of cell-surface secreted forms of the glyco-protein of vesicular stomatitis virus in eucaryotic cells. Cell 1982; 30(3):753–62.PubMedCrossRefGoogle Scholar
  10. 10.
    Gallione CJ, Rose JK. Nucleotide sequence of a cDNA clone encoding the entire glycoprotein from the New Jersey serotype of vesicular stomatitis virus. J Virol 1983; 46(1): 162–9.PubMedGoogle Scholar
  11. 11.
    Hirschberg K, Miller CM, Ellenberg J et al. Kinetic analysis of secretory protein traffic and characterization of Golgi to plasma membrane transport intermediates in living cells. J Cell Biol 1998; 143(6):1485–503.PubMedCrossRefGoogle Scholar
  12. 12.
    Esmon B, Novick P, Schekman R. Compartmentalized assembly of oligosaccharides on exported glycoproteins in yeast. Cell 1981; 25(2):451–60.PubMedCrossRefGoogle Scholar
  13. 13.
    Graham TR, Emr SD. Compartmental organization of Golgi-specific protein modification and vacuolar protein sorting events defined in a yeast sec 18 (NSF) mutant. J Cell Biol 1991; 114(2):207–18.PubMedCrossRefGoogle Scholar
  14. 14.
    Stevens T, Esmon B, Schekman R. Early stages in the yeast secretory pathway are required for transport of carboxypeptidase Y to the vacuole. Cell 1982; 30(2):439–48.PubMedCrossRefGoogle Scholar
  15. 15.
    Stoorvogel W. Analysis of the endocytic system by using horseradish peroxidase. Trends Cell Biol 1998; 8(12):503–5.PubMedCrossRefGoogle Scholar
  16. 16.
    Maxfield FR, McGraw TE. Endocytic recycling. Nat Rev Mol Cell Biol 2004; 5(2):121–32.PubMedCrossRefGoogle Scholar
  17. 17.
    Jeon H, Blacklow SC. Structure and physiologic function of the low-density lipoprotein receptor. Annu Rev Biochem 2005; 74:535–62.PubMedCrossRefGoogle Scholar
  18. 18.
    Qian ZM, Li H, Sun H et al. Targeted drug delivery via the transferrin receptor-mediated endocytosis pathway. Pharmacol Rev 2002; 54(4):561–87.PubMedCrossRefGoogle Scholar
  19. 19.
    Woods JW, Doriaux M, Farquhar MG. Transferrin receptors recycle to cis and middle as well as trans Golgi cisternae in Ig-secreting myeloma cells. J Cell Biol 1986; 103(1):277–86.PubMedCrossRefGoogle Scholar
  20. 20.
    Anderson RG, Brown MS, Beisiegel U et al. Surface distribution and recycling of the low density lipoprotein receptor as visualized with antireceptor antibodies. J Cell Biol 1982; 93(3):523–31.PubMedCrossRefGoogle Scholar
  21. 21.
    Sorkin A, Von Zastrow M. Signal transduction and endocytosis: Close encounters of many kinds. Nat Rev Mol Cell Biol 2002; 3(8):600–14.PubMedCrossRefGoogle Scholar
  22. 22.
    Marmor MD, Yarden Y. Role of protein ubiquitylation in regulating endocytosis of receptor tyrosine kinases. Oncogene 2004; 23(11):2057–70.PubMedCrossRefGoogle Scholar
  23. 23.
    Hicke L, Riezman H. Ubiquitination of a yeast plasma membrane receptor signals its ligand-stimulated endocytosis. Cell 1996; 84(2):277–87.PubMedCrossRefGoogle Scholar
  24. 24.
    Geli MI, Riezman H. Endocytic internalization in yeast and animal cells: Similar and different. J Cell Sci 1998; 111(Pt 8):1031–7.PubMedGoogle Scholar
  25. 25.
    Balch WE, Dunphy WG, Braell WA et al. Reconstitution of the transport of protein between successive compartments of the Golgi measured by the coupled incorporation of N-acetylglucosamine. Cell 1984; 39:405–16.PubMedCrossRefGoogle Scholar
  26. 26.
    Balch WE, Rothman JE. Characterization of protein transport between successive compartments of the Golgi apparatus: Asymmetric properties of donor and acceptor activities in a cell-free system. Arch Biochem Biophys 1985; 240:413–25.PubMedCrossRefGoogle Scholar
  27. 27.
    Balch WE, Glick BS, Rothman JE. Sequential intermediates in the pathway of intercompartmental transport in a cell-free system. Cell 1984; 39:525–36.PubMedCrossRefGoogle Scholar
  28. 28.
    Block MR, Glick BS, Wilcox CA et al. Purification of an N-ethylmaleimide-sensitive protein catalyzing vesicular transport. Proc Natl Acad Sci USA 1988; 85:7852–6.PubMedCrossRefGoogle Scholar
  29. 29.
    Orci L, Malhotra V, Amherdt M et al. Dissection of a single round of vesicular transport: Sequential coated and uncoated intermediates mediate intercisternal movement in the Golgi stack. Cell 1988; 56:357–8.CrossRefGoogle Scholar
  30. 30.
    Cook NR, Davidson HW. In vitro assays of vesicular transport. Traffic 2001; 2(1): 19–25.PubMedCrossRefGoogle Scholar
  31. 31.
    Rothman JE. Methods in Enzymology: Reconstitution of Intracellular Transport. Elsevier 1992; 219:1–438.CrossRefGoogle Scholar
  32. 32.
    Diaz R, Mayorga L, Stahl P. In vitro fusion of endosomes following receptor-mediated endocytosis. J Biol Chem 1988; 263(13):6093–100.PubMedGoogle Scholar
  33. 33.
    Gruenberg JE, Howell KE. Reconstitution of vesicle fusions occurring in endocytosis with a cell-free system. EMBO J 1986; 5(12):3091–101.PubMedGoogle Scholar
  34. 34.
    Veit B, Yucel JK, Malhotra V. Microtubule independent vesiculation of Golgi membranes and the reassembly of vesicles into Golgi stacks. J Cell Biol 1993; 122:1197–206.PubMedCrossRefGoogle Scholar
  35. 35.
    Rabouille C, Misteli T, Watson R et al. Reassembly of Golgi stacks from mitotic Golgi fragments in a cell-free system. J Cell Biol 1995; 129(3):605–18.PubMedCrossRefGoogle Scholar
  36. 36.
    Beckers DJM, Keller DS, Balch WE. Semi-intact cells permeable to macromolecules: Use in reconstitution of protein transport from the endoplasmic reticulum to the Golgi complex. Cell 1987; 50:523–34.PubMedCrossRefGoogle Scholar
  37. 37.
    Simons K, Virta H. Perforated MDCK cells support intracellular transport. EMBO J 1987; 6(8):2241–7.PubMedGoogle Scholar
  38. 38.
    Gravotta D, Adesnik M, Sabatini DD. Transport of influenza HA from the trans-Golgi network to the apical surface of MDCK cells permeabilized in their basolateral plasma membranes: Energy dependence and involvement of GTP-binding proteins. J Cell Biol 1990; 111(6 Pt 2):2893–908.PubMedCrossRefGoogle Scholar
  39. 39.
    Miller SG, Moore HP. Reconstitution of constitutive secretion using semi-intact cells: Regulation by GTP but not calcium. J Cell Biol 1991; 112(1):39–54.PubMedCrossRefGoogle Scholar
  40. 40.
    Braell WA. Detection of endocytic vesicle fusion in vitro, using assay based on avidin-biotin association reaction. Methods Enzymol 1992; 219:12–21.PubMedCrossRefGoogle Scholar
  41. 41.
    Mayer A, Wickner W. Docking of yeast vacuoles is catalyzed by the RAS-like GTPase YPT7P after symmetric priming by SEC18P (NSF). J Cell Biol 1997; 136(2):307–17.PubMedCrossRefGoogle Scholar
  42. 42.
    Misteli T, Warren G. A role for tubular networks and a COP I-independent pathway in the mitotic fragmentation of Golgi stacks in a cell-free system. J Cell Biol 1995; 130(5): 1027–39.PubMedCrossRefGoogle Scholar
  43. 43.
    Groesch ME, Ruohola H, Bacon R et al. Isolation of a functional vesicular intermediate that mediates ER to Golgi transport in yeast. J Cell Biol 1990; 111(1):45–53.PubMedCrossRefGoogle Scholar
  44. 44.
    Bennett MK, Wandinger-Ness A, Simons K. Release of putative exocytic transport vesicles from perforated MDCK cells. EMBO J 1988; 7(13):4075–85.Google Scholar
  45. 45.
    Salamero J, Stzul ES, Howell KE. Exocytic transport vesicles generated in vitro from the trans-Golgi network carry secretory and plasma membrane proteins. Proc Natl Acad Sci USA 1990; 87:7717–21.PubMedCrossRefGoogle Scholar
  46. 46.
    Martin TF, Walent JH. A new method for cell permeabilization reveals a cytosolic protein requirement for Ca2+-activated secretion in GH3 pituitary cells. J Biol Chem 1989; 264(17): 10299–308.PubMedGoogle Scholar
  47. 47.
    Clary DO, Rothman JE. Purification of three related peripheral membrane proteins needed for vesicular transport. J Biol Chem 1990; 265:10109–17.PubMedGoogle Scholar
  48. 48.
    Waters MG, Clary DO, Rothman JE. A novel 115-kD peripheral membrane protein is required for intercisternal transport in the Golgi stack. J Cell Biol 1992; 118:1015–26.PubMedCrossRefGoogle Scholar
  49. 49.
    Griff IC, Schekman R, Rothman JE et al. The yeast SEC 17 gene product is functionally equivalent to mammalian α-SNAP protein. J Biol Chem 1992; 267:12106–15.PubMedGoogle Scholar
  50. 50.
    Wilson DW, Wilcox CA, Flynn GC et al. A fusion protein required for vesicle-mediated transport in both mammalian cells and yeast. Nature 1989; 339:335–9.CrossRefGoogle Scholar
  51. 51.
    Baker D, Hicke L, Rexach M et al. Reconstitution of SEC gene product-dependent intercompartmental protein transport. Cell 1988; 54(3):335–44.PubMedCrossRefGoogle Scholar
  52. 52.
    Spang A, Schekman R. Reconstitution of retrograde transport from the Golgi to the ER in vitro. J Cell Biol 1998; 143(3):589–99.PubMedCrossRefGoogle Scholar
  53. 53.
    Conradt B, Haas A, Wickner W. Determination of four biochemically distinct, sequential stages during vacuole inheritance in vitro. J Cell Biol 1994; 126(1):99–110.PubMedCrossRefGoogle Scholar
  54. 54.
    Bonifacino JS, Glick BS. The mechanisms of vesicle budding and fusion. Cell 2004; 116(2):153–66.PubMedCrossRefGoogle Scholar
  55. 55.
    Haselbeck A, Schekman R. Interorganelle transfer and glycosylation of yeast invertase in vitro. Proc Natl Acad Sci USA 1986; 83(7):2017–21.PubMedCrossRefGoogle Scholar
  56. 56.
    Ruohola H, Kabcenell AK, Ferro-Novick S. Reconstitution of protein transport from the endoplasmic reticulum to the Golgi complex in yeast: The acceptor Golgi compartment is defective in the sec23 mutant. J Cell Biol 1988; 107(4): 1465–76.PubMedCrossRefGoogle Scholar
  57. 57.
    Muniz M, Martin ME, Hidalgo J et al. Protein kinase A activity is required for the budding of constitutive transport vesicles from the trans-Golgi network. Proc Natl Acad Sci USA 1997; 94(26):14461–6.PubMedCrossRefGoogle Scholar
  58. 58.
    Peter F, Wong SH, Subramaniam VN et al. Alpha-SNAP but not gamma-SNAP is required for ER-Golgi transport after vesicle budding and the Rabl-requiring step but before the EGTA-sensitive step._J Cell Sci 1998; 111(Part 17):2625–33.PubMedGoogle Scholar
  59. 59.
    Simon JP, Shen TH, Ivanov IE et al. Coatomer, but not P200 myosin II, is required for the in vitro formation of trans-Golgi network-derived vesicles containing the envelope glycoprotein of vesicular stomatitis virus. Proc Natl Acad Sci USA 1998; 95(3): 1073–8.PubMedCrossRefGoogle Scholar
  60. 60.
    Tang BL, Peter F, Krijnselocker J et al. The mammalian homolog of yeast Secl3p is enriched in the intermediate compartment and is essential for protein transport from the endoplasmic reticulum to the Golgi apparatus. Mol Cell Biol 1997; 17(1):256–66.PubMedGoogle Scholar
  61. 61.
    Taylor TC, Kanstein M, Weidman P et al. Cytosolic ARFs are required for vesicle formation but not for cell-free intra-Golgi transport: Evidence for coated vesicle-independent transport. Mol Biol Cell 1994; 5:237–52.PubMedGoogle Scholar
  62. 62.
    Sonnichsen B, Lowe M, Levine T et al. Role for giantin in docking COPI vesicles to Golgi membranes. J Cell Biol 1998; 140(5):1013–21.PubMedCrossRefGoogle Scholar
  63. 63.
    Kahn RA, Randazzo P, Serafini T et al. 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; 267:13039–46.PubMedGoogle Scholar
  64. 64.
    Plutner H, Schwaninger R, Pind S et al. Synthetic peptides of the Rab effector domain inhibit vesicular transport through the secretory pathway. EMBO J 1990; 9:2375–83.PubMedGoogle Scholar
  65. 65.
    GutiErrez LM, Chnaves JM, Ferrer-Montiel AV et al. A peptide that mimics the carboxy-terminal domain of SNAP-25 blocks Ca2+-dependent exocytosis in chromaffin cells. FEBS Lett 1995; 372(1):39–43.PubMedCrossRefGoogle Scholar
  66. 66.
    Brown WJ, Dewald DB, Emr SD et al. Role for phosphatidylinositol 3-kinase in the sorting and transport of newly synthesized lysosomal enzymes in mammalian cells. J Cell Biol 1995; 130(4):781–96.PubMedCrossRefGoogle Scholar
  67. 67.
    Taylor TC, Melancon P. ADP-ribosylation factor (ARF) mediates the effect of GTPyS on a cell-free intra-Golgi transport assay. J Cell Biol 1991; 115:245.CrossRefGoogle Scholar
  68. 68.
    Weidman PJ, Winter WM. The G protein-activating peptide, mastoparan, and the synthetic NH2-terminal ARF peptide, ARFpl3, inhibit in vitro Golgi transport by irreversibly damaging membranes. J Cell Biol 1994; 127:1815–27.PubMedCrossRefGoogle Scholar
  69. 69.
    Li L, Fleming N. Aluminum fluoride inhibits phospholipase D activation by a GTP-binding protein-independent mechanism. FEBS Lett 1999; 458(3):419–23.PubMedCrossRefGoogle Scholar
  70. 70.
    MacLean CM, Law GJ, Edwardson JM. Stimulation of exocytotic membrane fusion by modified peptides of the rab3 effector domain: Reevaluation of the role of rab3 in regulated exocytosis. Biochem J 1993; 294(Pt 2):325–8.PubMedGoogle Scholar
  71. 71.
    Fensome A, Cunningham E, Troung O et al. ARF 1(2–17) does not specifically interact with ARF 1-dependent pathways. Inhibition by peptide of phospholipases C beta, D and exocytosis in HL60 cells. FEBS Lett 1994; 349(1):34–8.PubMedCrossRefGoogle Scholar
  72. 72.
    Malhotra V, Orci L, Glick GS et al. Role of an N-ethylmaleimide-sensitive transport component in promoting fusion of transport vesicles with cisternae of the Golgi stack. Cell 1988; 54:221–7.PubMedCrossRefGoogle Scholar
  73. 73.
    Mayer A, Wickner W, Haas A. Sec18p (NSF)-driven release of sec17p (Alpha-SNAP) can precede docking and fusion of yeast vacuoles. Cell 1996; 85(1):83–94.PubMedCrossRefGoogle Scholar
  74. 74.
    Beckers CJM, Balch WE. Calcium and GTP: Essential components in vesicular trafficking between the endoplasmic reticulum and Golgi apparatus. J Cell Biol 1989; 108:1245–56.PubMedCrossRefGoogle Scholar
  75. 75.
    Wickner W. Yeast vacuoles and membrane fusion pathways. EMBO J 2002; 21(6): 1241–7.PubMedCrossRefGoogle Scholar
  76. 76.
    Takei K, Slepnev VI, Haucke V et al. Functional partnership between amphiphysin and dynamin in clathrin-mediated endocytosis. Nature Cell Biology 1999; 1(1):33–9.PubMedCrossRefGoogle Scholar
  77. 77.
    Zhu YX, Drake MT, Kornfeld S. ADP-ribosylation factor 1 dependent clathrin-coat assembly on synthetic liposomes. Proc Natl Acad Sci USA 1999; 96(9):5013–8.PubMedCrossRefGoogle Scholar
  78. 78.
    Drake MT, Zhu YX, Kornfeld S. The assembly of AP-3 adaptor complex-containing clathrin-coated vesicles on synthetic liposomes. Mol Biol Cell 2000; 11(11):3723–36.PubMedGoogle Scholar
  79. 79.
    Antonny B, Madden D, Hamamoto S et al. Dynamics of the COPII coat with GTP and stable analogues. Nat Cell Biol 2001; 3(6):531–7.PubMedCrossRefGoogle Scholar
  80. 80.
    Nickel W, Wieland FT. Receptor-dependent formation of COPI-coated vesicles from chemically defined donor liposomes [Review]. Regulators And Effectors Of Small Gtpases, Pt E: Gtpases Involved In Vesicular Traffic 2001; 329:388–404.CrossRefGoogle Scholar
  81. 81.
    Puertollano R, Randazzo PA, Presley JF et al. The GGAs promote ARF-dependent recruitment of clathrin to the TGN. Cell 2001; 105(1):93–102.PubMedCrossRefGoogle Scholar
  82. 82.
    Miller E, Antonny B, Hamamoto S et al. Cargo selection into COPII vesicles is driven by the Sec24p subunit. EMBO J 2002; 21(22):6105–13.PubMedCrossRefGoogle Scholar
  83. 83.
    Reinhard C, Schweikert M, Wieland FT et al. Functional reconstitution of COPI coat assembly and disassembly using chemically defined components. Proc Natl Acad Sci USA 2003; 100(14):8253–7.PubMedCrossRefGoogle Scholar
  84. 84.
    Sato K, Nakano A. Reconstitution of coat protein complex II (COPII) vesicle formation from cargo-reconstituted proteoliposomes reveals the potential role of GTP hydrolysis by Sarlp in protein sorting. J Biol Chem 2004; 279(2): 1330–5.PubMedCrossRefGoogle Scholar
  85. 85.
    Nickel W, Weber T, McNew JA et al. Content mixing and membrane integrity during membrane fusion driven by pairing of isolated v-SNAREs and t-SNAREs. Proc Natl Acad Sci USA 1999; 96(22):12571–6.PubMedCrossRefGoogle Scholar
  86. 86.
    Bai JH, Earles CA, Lewis JL et al. Membrane-embedded synaptotagmin penetrates cis or trans target membranes and clusters via a novel mechanism. J Biol Chem 2000; 275(33):25427–35.PubMedCrossRefGoogle Scholar
  87. 87.
    Parlati F, McNew JA, Fukuda R et al. Topological restriction of SNARE-dependent membrane fusion. Nature 2000; 407(6801): 194–8.PubMedCrossRefGoogle Scholar
  88. 88.
    de Haro L, Ferracci G, Opi S et al. Ca2+/calmodulin transfers the membrane-proximal lipid-binding domain of the v-SNARE synaptobrevin from cis to trans bilayers. Proc Natl Acad Sci USA 2004; 101(6):1578–83.PubMedCrossRefGoogle Scholar
  89. 89.
    Schuette CG, Hatsuzawa K, Margittai M et al. Determinants of liposome fusion mediated by synaptic SNARE proteins. Proc Natl Acad Sci USA 2004; 101(9):2858–63.PubMedCrossRefGoogle Scholar
  90. 90.
    Bowen ME, Weninger K, Brunger AT et al. Single molecule observation of liposome-bilayer fusion thermally induced by soluble N-ethyl maleimide sensitive-factor attachment protein receptors (SNAREs). Biophys J 2004; 87(5):3569–84.PubMedCrossRefGoogle Scholar
  91. 91.
    Tucker WC, Weber T, Chapman ER. Reconstitution of Ca2+-regulated membrane fusion by synaptotagmin and SNAREs. Science 2004; 304(5669):435–8.PubMedCrossRefGoogle Scholar
  92. 92.
    Zhang Y, Su Z, Zhang F et al. A partially zipped SNARE complex stabilized by the membrane. J Biol Chem 2005; 280(16): 15595–600.PubMedCrossRefGoogle Scholar
  93. 93.
    Lu X, Zhang F, McNew JA et al. Membrane fusion induced by neuronal SNAREs transits through hemifusion. J Biol Chem 2005; 280(34):30538–41.PubMedCrossRefGoogle Scholar
  94. 94.
    Liu T, Tucker WC, Bhalla A et al. SNARE-driven, 25-millisecond vesicle fusion in vitro. Biophys J 2005; 89(4):2458–72.PubMedCrossRefGoogle Scholar
  95. 95.
    Xu Y, Zhang F, Su Z et al. Hemifusion in SNARE-mediated membrane fusion. Nat Struct Mol Biol 2005; 12(5):417–22.PubMedCrossRefGoogle Scholar
  96. 96.
    Lu X, Xu Y, Zhang F et al. Synaptotagmin I and Ca(2+) promote half fusion more than full fusion in SNARE-mediated bilayer fusion. FEBS Lett 2006; 580(9):2238–46.PubMedCrossRefGoogle Scholar
  97. 97.
    Fix M, Melia TJ, Jaiswal JK et al. Imaging single membrane fusion events mediated by SNARE proteins. Proc Natl Acad Sci USA 2004; 101(19):7311–6.PubMedCrossRefGoogle Scholar
  98. 98.
    Antonny B, Bigay J, Casella JF et al. Membrane curvature and the control of GTP hydrolysis in Arfl during COPI vesicle formation. Biochem Soc Trans 2005; 33(Pt 4):619–22.PubMedCrossRefGoogle Scholar
  99. 99.
    Bielli A, Haney CJ, Gabreski G et al. Regulation of Sari NH2 terminus by GTP binding and hydrolysis promotes membrane deformation to control COPII vesicle fission. J Cell Biol 2005; 171(6):919–24.PubMedCrossRefGoogle Scholar
  100. 100.
    Bigay J, Gounon P, Robineau S et al. Lipid packing sensed by ArfGAPl couples COPI coat disassembly to membrane bilayer curvature. Nature 2003; 426(6966):563–6.PubMedCrossRefGoogle Scholar
  101. 101.
    Lee MC, Orci L, Hamamoto S et al. Sarlp N-terminal helix initiates membrane curvature and completes the fission of a COPII vesicle. Cell 2005; 122(4):605–17.PubMedCrossRefGoogle Scholar
  102. 102.
    Matsuo H, Chevallier J, Mayran N et al. Role of LBPA and Alix in multivesicular liposome formation and endosome organization. Science 2004; 303(5657):531–4.PubMedCrossRefGoogle Scholar
  103. 103.
    Peter BJ, Kent HM, Mills IG et al. BAR domains as sensors of membrane curvature: The amphiphysin BAR structure. Science 2004; 303(5657):495–9.PubMedCrossRefGoogle Scholar
  104. 104.
    Roux A, Cappello G, Cartaud J et al. A minimal system allowing tubulation with molecular motors pulling on giant liposomes. Proc Natl Acad Sci USA 2002; 99(8):5394–9.PubMedCrossRefGoogle Scholar
  105. 105.
    Yoshida Y, Kinuta M, Abe T et al. The stimulatory action of amphiphysin on dynamin function is dependent on lipid bilayer curvature. EMBO J 2004; 23(17):3483–91.PubMedCrossRefGoogle Scholar
  106. 106.
    Ferro-Novick S, Novick P, Field C et al. Yeast secretory mutants that block the formation of active cell surface enzymes. J Cell Biol 1984; 98(1):35–43.PubMedCrossRefGoogle Scholar
  107. 107.
    Novick P, Field C, Schekman R. Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway. Cell 1980; 21(1):205–15.PubMedCrossRefGoogle Scholar
  108. 108.
    Ferro-Novick S, Jahn R. Vesicle fusion from yeast to man. Nature 1994; 370:191–3.PubMedCrossRefGoogle Scholar
  109. 109.
    Franzusoff A. Beauty and the yeast: Compartmental organization of the secretory pathway. Semin Cell Biol 1992; 3(5):309–24.PubMedCrossRefGoogle Scholar
  110. 110.
    Schekman R, Novick P. 23 genes, 23 years later. Cell 2004; 116(2 Suppl):S13–5, (1 p following S19).CrossRefGoogle Scholar
  111. 111.
    Bankaitis VA, Johnson LM, Emr SD. Isolation of yeast mutants defective in protein targeting to the vacuole. Proc Natl Acad Sci USA 1986; 83(23):9075–9.PubMedCrossRefGoogle Scholar
  112. 112.
    Rothman JH, Stevens TH. Protein sorting in yeast: Mutants defective in vacuole biogenesis mislocalize vacuolar proteins into the late secretory pathway. Cell 1986; 47(6): 1041–51.PubMedCrossRefGoogle Scholar
  113. 113.
    Banta LM, Robinson JS, Klionsky DJ et al. Organelle assembly in yeast: Characterization of yeast mutants defective in vacuolar biogenesis and protein sorting. J Cell Biol 1988; 107(4): 1369–83.PubMedCrossRefGoogle Scholar
  114. 114.
    Vida TA, Emr SD. A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast. J Cell Biol 1995; 128(5):779–92.PubMedCrossRefGoogle Scholar
  115. 115.
    Robinson JS, Graham TR, Emr SD. A putative zinc finger protein, Saccharomyces cerevisiae Vpsl8p, affects late Golgi functions required for vacuolar protein sorting and efficient alpha-factor prohormone maturation. Mol Cell Biol 1991; ll(12):5813–24.Google Scholar
  116. 116.
    Kaiser CA, Schekman R. Distinct sets of SEC genes govern transport vesicle formation and fusion early in the secretory pathway. Cell 1990; 61:723–33.PubMedCrossRefGoogle Scholar
  117. 117.
    Rieder SE, Emr SD. A novel ring finger protein complex essential for a late step in protein transport to the yeast vacuole. Mol Biol Cell 1997; 8(ll):2307–27.PubMedGoogle Scholar
  118. 118.
    Prescianottobaschong C, Riezman H. Morphology of the yeast endocytic pathway. Mol Biol Cell 1998; 9(l):173–89.Google Scholar
  119. 119.
    Raymond CK, Howald-Stevenson I, Vater CA et al. Morphological classification of the yeast vacuola protein sorting mutants: Evidence for a prevacuolar compartment in class E vps mutants. Mol Biol Cell 1992; 3(12):1389–402.PubMedGoogle Scholar
  120. 120.
    Rothman JH, Howald I, Stevens TH. Characterization of genes required for protein sorting and vacuolar function in the yeast Saccharomyces cerevisiae. EMBO J 1989; 8(7):2057–65.PubMedGoogle Scholar
  121. 121.
    Sciorra VA, Audhya A, Parsons AB et al. Synthetic genetic array analysis of the Ptdlns 4-kinase Piklp identifies components in a Golgi-specific Ypt31/rab-GTPase signaling pathway. Mol Biol Cell 2005; 16(2):776–93.PubMedCrossRefGoogle Scholar
  122. 122.
    Singer-Kruger B, Stenmark H, Zerial M. Yeast Ypt51p and mammalian Rab5: Counterparts with similar function in the early endocytic pathway. J Cell Sci 1995; 108(Pt 11):3509–21.PubMedGoogle Scholar
  123. 123.
    Zerial M, McBride H. Rab proteins as membrane organizers. Nat Rev Mol Cell Biol 2001; 2(2):107–17.PubMedCrossRefGoogle Scholar
  124. 124.
    Giaever G, Chu AM, Ni L et al. Functional profiling of the Saccharomyces cerevisiae genome. Nature 2002; 418(6896):387–91.PubMedCrossRefGoogle Scholar
  125. 125.
    Ooi SL, Pan X, Peyser BD et al. Global synthetic-lethality analysis and yeast functional profiling. Trends Genet 2006; 22(l):56–63.PubMedCrossRefGoogle Scholar
  126. 126.
    Allen RD, Brown DT, Gilbert SP et al. Transport of vesicles along filaments dissociated from squid axoplasm. Biol Bull 1983; 165:523.Google Scholar
  127. 127.
    Hayden JH, Allen RD, Goldman RD. Cytoplasmic transport in keratocytes: Direct visualization of particle translocation along microtubules. Cell Motil 1983; 3(1): 1–19.CrossRefGoogle Scholar
  128. 128.
    Vale RD, Reese TS, Sheetz MP. Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility. Cell 1985; 42(l):39–50.PubMedCrossRefGoogle Scholar
  129. 129.
    Presley JF, Cole NB, Schroer TA et al. ER-to-Golgi transport visualized in living cells. Nature 1997; 389(6646):81–5.PubMedCrossRefGoogle Scholar
  130. 130.
    Jones AT, Clague MJ. Phosphatidylinositol 3-kinase activity is required for early endosome fusion. Biochem J 1995; 311(Pt l):31–4.PubMedGoogle Scholar
  131. 131.
    Li G, D’Souza-Schorey C, Barbieri MA et al. Evidence for phosphatidylinositol 3-kinase as a regulator of endocytosis via activation of Rab5. Proc Natl Acad Sci USA 1995; 92(22): 10207–11.PubMedCrossRefGoogle Scholar
  132. 132.
    Spiro DJ, Boll W, Kirchhausen T et al. Wortmannin alters the transferrin receptor endocytic pathway in vivo and in vitro. Mol Biol Cell 1996; 7(3):355–67.PubMedGoogle Scholar
  133. 133.
    Nakanishi S, Catt KJ, Balla T. A wortmannin-sensitive phosphatidylinositol 4-kinase that regulates hormone-sensitive pools of inositolphospholipids. Proc Natl Acad Sci USA 1995; 92(12):5317–21.PubMedCrossRefGoogle Scholar
  134. 134.
    Preuss ML, Schmitz AJ, Thole JM et al. A role for the RabA4b effector protein PI-4Ktal in polarized expansion of root hair cells in Arabidopsis thaliana. J Cell Biol 2006; 172(7):991–8.Google Scholar
  135. 135.
    Bomsel M, Prydz K, Parton RG et al. Endocytosis in filter-grown Madin-Darby canine kidney cells. J Cell Biol 1989; 109(6 Pt 2):3243–58.PubMedCrossRefGoogle Scholar
  136. 136.
    Murphy RF. Analysis and isolation of endocytic vesicles by flow cytometry and sorting: Demonstration of three kinetically distinct compartments involved in fluid-phase endocytosis. Proc Natl Acad Sci USA 1985; 82(24):8523–6.PubMedCrossRefGoogle Scholar
  137. 137.
    Merion M, Schlesinger P, Brooks RM et al. Defective acidification of endosomes in Chinese hamster ovary cell mutants “cross-resistant” to toxins and viruses. Proc Natl Acad Sci USA 1983; 80(17):5315–9.PubMedCrossRefGoogle Scholar
  138. 138.
    Shurety W, Stewart NL, Stow JL et al. Fluid-phase markers in the basolateral endocytic pathway accumulate in response to the actin assembly-promoting drug Jasplakinolide. Mol Biol Cell 1998; 9(4):957–75.PubMedGoogle Scholar
  139. 139.
    von Bonsdorff CH, Fuller SD, Simons K. Apical and basolateral endocytosis in Madin-Darby canine kidney (MDCK) cells grown on nitrocellulose filters. EMBO J 1985; 4(ll):2781–92.Google Scholar
  140. 140.
    Walter RJ, Berlin RD, Pfeiffer JR et al. Polarization of endocytosis and receptor topography on cultured macrophages. J Cell Biol 1980; 86(1):199–211.PubMedCrossRefGoogle Scholar
  141. 141.
    Schmid SL, Fuchs R, Male P et al. Two distinct subpopulations of endosomes involved in membrane recycling and transport to lysosomes. Cell 1988; 52(l):73–83.PubMedCrossRefGoogle Scholar
  142. 142.
    Betz WJ, Mao F, Smith CB. Imaging exocytosis and endocytosis. Curr Opin Neurobiol 1996; 6(3):365–71.PubMedCrossRefGoogle Scholar
  143. 143.
    Cochilla AJ, Angleson JK, Betz WJ. Monitoring secretory membrane with FM1-43 fluorescence. Annu Rev Neurosci 1999; 22:1–10.PubMedCrossRefGoogle Scholar
  144. 144.
    Vida TA, Emr SD. A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast. J Cell Biol 1995; 128(5):779–92.PubMedCrossRefGoogle Scholar
  145. 145.
    Prasher DC, Eckenrode VK, Ward WW et al. Primary structure of the Aequorea victoria green-fluorescent protein. Gene 1992; lll(2):229–33.CrossRefGoogle Scholar
  146. 146.
    Cubitt AB, Heim R, Adams SR et al. Understanding, improving and using green fluorescent proteins. Trends Biochem Sci 1995; 20(ll):448–55.PubMedCrossRefGoogle Scholar
  147. 147.
    Heim R, Cubitt AB, Tsien RY. Improved green fluorescence. Nature 1995; 373(6516):663–4.PubMedCrossRefGoogle Scholar
  148. 148.
    Heim R, Prasher DC, Tsien RY. Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc Natl Acad Sci USA 1994; 91(26):12501–4.PubMedCrossRefGoogle Scholar
  149. 149.
    Heim R, Tsien RY. Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr Biol 1996; 6(2): 178–82.PubMedCrossRefGoogle Scholar
  150. 150.
    Cormack BP, Valdivia RH, Falkow S. FACS-optimized mutants of the green fluorescent protein (GFP). Gene 1996; 173(1 Spec No):33–8.PubMedCrossRefGoogle Scholar
  151. 151.
    Tsien RY. The green fluorescent protein. Annu Rev Biochem 1998; 67:509–44.PubMedCrossRefGoogle Scholar
  152. 152.
    Wang S, Hazelrigg T. Implications for bed mRNA localization from spatial distribution of exu protein in Drosophila oogenesis. Nature 1994; 369(6479):400–3.PubMedCrossRefGoogle Scholar
  153. 153.
    Rizzuto R, Brini M, Pizzo P et al. Chimeric green fluorescent protein as a tool for visualizing subcellular organelles in living cells. Curr Biol 1995; 5(6):635–42.PubMedCrossRefGoogle Scholar
  154. 154.
    Kaether C, Gerdes HH. Visualization of protein transport along the secretory pathway using green fluorescent protein. FEBS Lett 1995; 369(2–3):267–71.PubMedCrossRefGoogle Scholar
  155. 155.
    Hampton RY, Koning A, Wright R et al. In vivo examination of membrane protein localization and degradation with green fluorescent protein. Proc Natl Acad Sci USA 1996; 93(2):828–33.PubMedCrossRefGoogle Scholar
  156. 156.
    Scales SJ, Pepperkok R, Kreis TE. Visualization of ER-to-Golgi transport in living cells reveals a sequential mode of action for COPII and COPI. Cell 1997; 90(6): 1137–48.PubMedCrossRefGoogle Scholar
  157. 157.
    Storrie B, White J, Rottger S et al. Recycling of golgi-resident glycosyltransferases through the ER reveals a novel pathway and provides an explanation for nocodazole-induced Golgi scattering. J Cell Biol 1998; 143(6):1505–21.PubMedCrossRefGoogle Scholar
  158. 158.
    Hirschberg K, Miller CM, Ellenberg J et al. Kinetic analysis of secretory protein traffic and characterization of golgi to plasma membrane transport intermediates in living cells. J Cell Biol 1998; 143(6):1485–503.PubMedCrossRefGoogle Scholar
  159. 159.
    Bergmann JE. Using temperature-sensitive mutants of VSV to study membrane protein biogenesis. Methods Cell Biol 1989; 32:85–110.PubMedCrossRefGoogle Scholar
  160. 160.
    Poo M, Cone RA. Lateral diffusion of rhodopsin in the photoreceptor membrane. Nature 1974; 247(441):438–41.PubMedCrossRefGoogle Scholar
  161. 161.
    Peters R, Peters J, Tews KH et al. A microfluorimetric study of translational diffusion in erythrocyte membranes. Biochim Biophys Acta 1974; 367(3):282–94.PubMedCrossRefGoogle Scholar
  162. 162.
    Edidin M, Zagyansky Y, Lardner TJ. Measurement of membrane protein lateral diffusion in single cells. Science 1976; 191(4226):466–8.PubMedCrossRefGoogle Scholar
  163. 163.
    Jacobson K, Derzko Z, Wu ES et al. Measurement of the lateral mobility of cell surface components in single, living cells by fluorescence recovery after photobleaching. J Supramol Struct 1976; 5(4):565(4l7)–576(428).CrossRefGoogle Scholar
  164. 164.
    Schlessinger J, Koppel DE, Axelrod D et al. Lateral transport on cell membranes: Mobility of concanavalin A receptors on myoblasts. Proc Natl Acad Sci USA 1976; 73(7):2409–13.PubMedCrossRefGoogle Scholar
  165. 165.
    Ellenberg J, Siggia ED, Moreira JE et al. Nuclear membrane dynamics and reassembly in living cells: Targeting of an inner nuclear membrane protein in interphase and mitosis. J Cell Biol 1997; 138(6):1193–206.PubMedCrossRefGoogle Scholar
  166. 166.
    Cole NB, Sciaky N, Marotta A et al. Golgi dispersal during microtubule disruption: Regeneration of Golgi stacks at peripheral endoplasmic reticulum exit sites. Mol Biol Cell 1996; 7(4):631–50.PubMedGoogle Scholar
  167. 167.
    Nehls S, Snapp EL, Cole NB et al. Dynamics and retention of misfolded proteins in native ER membranes. Nat Cell Biol 2000; 2(5):288–95.PubMedCrossRefGoogle Scholar
  168. 168.
    Snapp EL, Hegde RS, Francolini M et al. Formation of stacked ER cisternae by low affinity protein interactions. J Cell Biol 2003; l63(2):257–69.CrossRefGoogle Scholar
  169. 169.
    Zaal KJ, Smith CL, Polishchuk RS et al. Golgi membranes are absorbed into and reemerge from the ER during mitosis. Cell 1999; 99(6):589–601.PubMedCrossRefGoogle Scholar
  170. 170.
    Ward TH, Polishchuk RS, Caplan S et al. Maintenance of Golgi structure and function depends on the integrity of ER export. J Cell Biol 2001; 155(4):557–70.PubMedCrossRefGoogle Scholar
  171. 171.
    Matz MV, Fradkov AF, Labas YA et al. Fluorescent proteins from nonbioluminescent Anthozoa species. Nat Biotechnol 1999; 17(10):969–73.PubMedCrossRefGoogle Scholar
  172. 172.
    Rizzuto R, Brini M, De Giorgi F et al. Double labelling of subcellular structures with organelle-targeted GFP mutants in vivo. Curr Biol 1996; 6(2): 183–8.PubMedCrossRefGoogle Scholar
  173. 173.
    Majoul I, Straub M, Hell SW et al. KDEL-cargo regulates interactions between proteins involved in COPI vesicle traffic: Measurements in living cells using FRET. Dev Cell 2001; l(l):139–53.CrossRefGoogle Scholar
  174. 174.
    Sorkina T, Doolen S, Galperin E et al. Oligomerization of dopamine transporters visualized in living cells by fluorescence resonance energy transfer microscopy. J Biol Chem 2003; 278(30):28274–83.PubMedCrossRefGoogle Scholar
  175. 175.
    Floyd DH, Geva A, Bruinsma SP et al. C5a receptor oligomerization. II. Fluorescence resonance energy transfer studies of a human G protein-coupled receptor expressed in yeast. J Biol Chem 2003; 278(37):35354–61.PubMedCrossRefGoogle Scholar
  176. 176.
    Giraudo CG, Maccioni HJ. Ganglioside glycosyltransferases organize in distinct multienzyme complexes in CHO-K1 cells. J Biol Chem 2003; 278(41):40262–71.PubMedCrossRefGoogle Scholar
  177. 177.
    Miranda M, Sorkina T, Grammatopoulos TN et al. Multiple molecular determinants in the carboxyl terminus regulate dopamine transporter export from endoplasmic reticulum. J Biol Chem 2004; 279(29):30760–70.PubMedCrossRefGoogle Scholar
  178. 178.
    Sato K, Nakano A. Dissection of COPII subunit-cargo assembly and disassembly kinetics during Sarlp-GTP hydrolysis. Nat Struct Mol Biol 2005; 12(2):167–74.PubMedCrossRefGoogle Scholar
  179. 179.
    Ormo M, Cubitt AB, Kallio K et al. Crystal structure of the Aequorea victoria green fluorescent protein. Science 1996; 273(5280): 1392–5.PubMedCrossRefGoogle Scholar
  180. 180.
    Goda Y, Pfeffer SR. Selective recycling of the mannose 6-phosphate/IGF-II receptor to the trans Golgi network in vitro. Cell 1988; 55:309–20.PubMedCrossRefGoogle Scholar
  181. 181.
    Vida TA, Graham TR, Emr SD. In vitro reconstitution of intercompartmental protein transport to the yeast vacuole. J Cell Biol 1990; 111(6 Pt 2):2871–84.PubMedCrossRefGoogle Scholar
  182. 182.
    Wessling-Resnick M, Braell WA. The sorting and segregation mechanism of the endocytic pathway is functional in a cell-free system. J Biol Chem 1990; 265(2):690–9.Google Scholar
  183. 183.
    Gruenberg J, Griffiths G, Howell KE. Characterization of the early endosome and putative endocytic carrier vesicles in vivo and with an assay of vesicle fusion in vitro. J Cell Biol 1989; 108(4): 1301–16.PubMedCrossRefGoogle Scholar
  184. 184.
    Mullock BM, Branch WJ, van Schaik M et al. Reconstitution of an endosome-lysosome interaction in a cell-free system. J Cell Biol 1989; 108(6):2093–9.PubMedCrossRefGoogle Scholar
  185. 185.
    Conradt B, Shaw J, Vida T et al. In vitro reactions of vacuole inheritance in Saccharomyces cerevisiae. J Cell Biol 1992; 119(6): 1469–79.PubMedCrossRefGoogle Scholar

Copyright information

© Landes Bioscience and Springer Science+Business Media 2009

Authors and Affiliations

  1. 1.Department of Molecular, Cellular & Developmental BiologyUniversity of MichiganAnn ArborUSA
  2. 2.Donald Danforth Plant Science CenterSt. LouisUSA
  3. 3.Office of Scientific ReviewNational Institute of General Medical SciencesBethesdaUSA

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