Structural insights into the molecular mechanism of Ca2+-dependent exocytosis

Current Opinion in Neurobiology

Volumn 10, Issue 3, 2000, Pages 293-302

  Brunger, Axel T ^a  

^a YALE UNIVERSITY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

CALCIUM ION; GUANINE NUCLEOTIDE BINDING PROTEIN; SNARE PROTEIN; SYNAPTOBREVIN; SYNAPTOTAGMIN; SYNTAXIN;

ANIMAL CELL; CALCIUM TRANSPORT; COMPLEX FORMATION; CRYSTAL STRUCTURE; EXOCYTOSIS; HUMAN; HUMAN CELL; MEMBRANE FUSION; NONHUMAN; PRIORITY JOURNAL; PROTEIN LIPID INTERACTION; PROTEIN PROTEIN INTERACTION; REVIEW; SYNAPSE VESICLE;

EID: 0034094172     PISSN: 09594388     EISSN: None     Source Type: Journal    
DOI: 10.1016/S0959-4388(00)00098-2     Document Type: Review

References (105)

1. Rothman J.E. Mechanisms of intracellular protein transport. Nature. 372:1994;55-63.
2. Südhof T.C. The synaptic vesicle cycle: a cascade of protein-protein interactions. Nature. 375:1995;645-653.
3. Schekman R., Orci L. Coat proteins and vesicle budding. Science. 271:1996;1526-1533.
4. Hanson P.I., Roth R., Morisaki H., Jahn R., Heuser J.E. Structure and conformational changes in NSF and its membrane receptor complexes visualized by quick-freeze/deep-etch electron microscopy. Cell. 90:1997;523-535.
5. Conner S., Wessel G.M. Rab3 mediates cortical granule exocytosis in the sea urchin egg. Dev Biol. 203:1998;334-344.
6. Bock J.B., Scheller R.H. SNARE proteins mediate lipid bilayer fusion. Proc Natl Acad Sci USA. 96:1999;12227-12229. The authors present results supporting the model that SNAREs are important in mediating membrane fusion.
7. Ferro-Novick S., Jahn R. Vesicle fusion from yeast to man. Nature. 370:1994;191-193.
8. Bennett M.K., Scheller R.H. The molecular machinery for secretion is conserved from yeast to neurons. Proc Natl Acad Sci USA. 90:1993;2559-2563.
9. Whiteheart S.W., Rossnagel K., Buhrow S.A., Brunner M., Jaenicke R., Rothman J.E. N-ethymaleimide-sensitive fusion protein: a trimeric ATPase whose hydrolysis of ATP is required for membrane fusion. J Cell Biol. 126:1994;945-954.
10. Clary D.O., Griff I.C., Rothman J.E. SNAPs, a family of NSF attachment proteins involved in intracellular membrane fusion in animals and yeast. Cell. 61:1990;709-721.
11. Ferro-Novick S., Novick P. The role of GTP-binding proteins in transport along the exocytic pathway. Annu Rev Cell Biol. 9:1993;575-599.
12. Brose N., Petrenko A.G., Südhof T.C., Jahn R. Synaptotagmin: a calcium sensor on the synaptic vesicle surface. Science. 256:1992;1021-1025.
13. Pevsner J., Hsu S-C., Scheller R.H. n-Sec1: a neuronal-specific syntaxin-binding protein. Proc Natl Acad Sci USA. 91:1994;1445-1449.
14. McMahon H.T., Missler M., Li C., Südhof T.C. Complexins: cytosolic proteins that regulate SNAP receptor function. Cell. 83:1995;111-119.
15. Skehel P.A., Armitage B.A., Bartsch D., Hu Y., Kaang B.K., Siegelbaum S.A., Kandel E.R., Martin K.C. Proteins functioning in synaptic transmission at the sensory to motor synapse of Aplysia. Neuropharmacology. 34:1995;1379-1385.
16. Wiedenmann B., Franke W.W. Identification and localization of synaptophysin, an integral membrane glycoprotein of Mr 38,000 characteristic of presynaptic vesicles. Cell. 41:1985;1017-1028.
17. Misura K.M.S., Scheller R.H., Weis W.I. Three-dimensional structure of the neuronal-Sec1/syntaxin 1a complex. Nature. 404:2000;355-362. The structure of the nSec1-syntaxin complex is presented. The conformation of syntaxin is dramatically different from its conformation when it forms part of the SNARE complex. A possible role of Rab proteins is proposed for dissociating the stable nSec1-syntaxin complex.
18. Hsu S.C., Ting A.E., Hazuka C.D., Davanger S., Kenny J.W., Kee Y., Scheller R.H. The mammalian brain rsec6/8 complex. Neuron. 17:1996;1209-1219.
19. TerBush D.R., Maurice T., Roth D., Novick P. The exocyst is a multiprotein complex required for exocytosis in Saccharomyces cerevisiae. EMBO J. 15:1996;6483-6494.
20. Otto H., Hanson P.I., Jahn R. Assembly and disassembly of a ternary complex of synaptobrevin, syntaxin, and SNAP-25 in the membrane of synaptic vesicles. Proc Natl Acad Sci USA. 94:1997;6197-6201.
21. Fasshauer D., Otto H., Eliason W.K., Jahn R., Brunger A.T. Structural changes are associated with SNARE-complex formation. J Biol Chem. 242:1997;28036-28041.
22. Rice L.M., Brennwald P., Brunger A.T. Formation of a yeast SNARE complex is accompanied by significant structural changes. FEBS Lett. 415:1997;49-55.
23. Poirier M.A., Hao J.C., Malkus P.N., Chan C., Moore M.F., King D.S., Bennett M.K. Protease resistance of syntaxin.SNAP-25.VAMP complexes. Implications for assembly and structure. J Biol Chem. 273:1998;11370-11377.
24. Fasshauer D., Eliason W.K., Brunger A.T., Jahn R. Identification of a minimal core of the synaptic SNARE complex sufficient for reversible assembly and disassembly. Biochemistry. 37:1998;10345-10353.
25. Parlatti F., Weber T., McNew J.A., Westermann B., Söllner T.H., Rothman J.E. Rapid and efficient fusion of phospholipid vesicles by the α-helical core of a SNARE complex in the absence of an N-terminal regulatory domain. Proc Natl Acad Sci USA. 96:1999;12565-12570. The authors show that the core of the SNARE complex (i.e. without the amino-terminal domain of syntaxin and the SNAP-25 linker region) is sufficient to promote vesicle fusion in vitro.
26. Sutton R.B., Fasshauer D., Jahn R., Brunger A.T. Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 Å resolution. Nature. 395:1998;347-353.
27. Fernandez I., Ubach J., Dulubova I., Zhang X., Südhof T.C., Rizo J. Three-dimensional structure of an evolutionarily conserved N-terminal domain of syntaxin 1A. Cell. 94:1998;841-849.
28. Fasshauer D., Sutton R.B., Brunger A.T., Jahn R. Conserved structural features of the synaptic fusion complex: SNARE proteins reclassified as Q- and R-SNAREs. Proc Natl Acad of Sci USA. 95:1998;15781-15786.
29. Ungermann C., von Mollard G.F., Jensen O.N., Margolis N., Stevens T.H., Wickner W. Three v-SNAREs and two t-SNAREs, present in a pentameric cis-SNARE complex on isolated vacuoles, are essential for homotypic fusion. J Cell Biol. 28:1999;1435-1442. Experiments using yeast extracts and immuno-blotting show that five SNARE proteins are involved in homotypic vesicle fusion. These experiments indicate interactions between the five yeast SNARE proteins. However, they do not conclusively show the existence of a pentameric SNARE complex.
30. 15N HSQC (heteronuclear single quantum correlation) NMR spectroscopy. Little structure is induced in Sso1 upon binary complex formation with Sec9p (SNAP-25 homologue). Upon ternary complex formation, both Sso1 and Snc1 become nearly entirely structured.
31. ••].
32. ••].
33. Nicholson K.L., Munson M., Miller R.B., Filip T.J., Fairman R., Hughson F.M. Regulation of SNARE complex assembly by an N-terminal domain of the t-SNARE Sso1p. Nat Struct Biol. 5:1998;793-802.
34. Fasshauer D., Bruns D., Shen B., Jahn R., Brunger A.T. A structural change occurs upon binding of syntaxin to SNAP-25. J Biol Chem. 272:1997;4582-4590.
35. Lin R.C., Scheller R.H. Structural organization of the synaptic exocytosis core complex. Neuron. 19:1997;1087-1094.
36. Hanson P.I., Heuser J.E., Jahn R. Neurotransmitter release - four years of SNARE complexes. Curr Opin Neurobiol. 7:1997;310-315.
37. Katz L., Hanson P.I., Heuser J.E., Brennwald P. Genetic and morphological analyses reveal a critical interaction between the C-termini of two SNARE proteins and a parallel four helical arrangement for the exocytic SNARE complex. EMBO J. 17:1998;6200-6209.
38. Poirier M.A., Xiao W., Macosko J.C., Chan C., Shin Y.K., Bennett M.K. The synaptic SNARE complex is a parallel four-stranded helical bundle. Nat Struct Biol. 5:1998;765-769.
39. Hayashi T., McMahon H., Yamasaki S., Binz T., Hata Y., Südhof T.C., Niemann H. Synaptic vesicle membrane fusion complex: action of clostridial neurotoxins on assembly. EMBO J. 13:1994;5051-5061.
40. Jahn R., Niemann H. Molecular mechanisms of clostridial neurotoxins. Ann NY Acad Sci. 733:1994;245-255.
41. Weber T., Zemelman B.V., McNew J.A., Westermann B., Gmachl M., Parlatti F., Söllner T.H., Rothman J.E. SNAREpins, minimal machinery for membrane fusion. Cell. 92:1998;759-772.
42. Nickel W., Weber T., McNew J.A., Parlatti F., Söllner T.H., Rothman J.E. Content mixing and membrane integrity during membrane fusion driven by pairing of isolated v-SNAREs and t-SNAREs. Proc Natl Acad Sci USA. 96:1999;12571-12576. Vesicle contents mixing assays illustrate the role of SNAREs in vesicle fusion using an in vitro liposome fusion system.
43. 2+ concentration increase.
44. Skehel J.J., Wiley D.C. Coiled coils in both intracellular vesicle and viral membrane fusion. Cell. 95:1998;871-874.
45. Hua S-H., Charlton M.P. Activity-dependent changes in partial VAMP complexes during neurotransmitter release. Nat Neurosci. 2:1999;1078-1083. Proteolysis by synaptobrevin-specific neurotoxins shows that the carboxy-terminal half of the SNARE core complex is exposed before neurotransmission. This suggests the existence of a partially assembled SNARE complex.
46. Xu T., Binz T., Niemann H., Neher E. Multiple kinetic components of exocytosis distinguished by neurotoxin sensitivity. Nat Neurosci. 1:1998;192-200.
47. 2+ leads to an exocytotic burst followed by sustained secretion. In the presence of an antibody that blocks SNARE complex assembly, the sustained component is largely blocked, the burst slightly reduced, and one of the kinetic components of the burst is eliminated. This suggests that SNARE complexes exist in a dynamic equilibrium between loose and tight states, both of which support exocytosis.
48. Lee J., Lentz B.R. Evolution of lipidic structures during model membrane fusion and the relation of this process to cell membrane fusion. Biochemistry. 36:1997;6251-6259.
49. Lee J., Lentz B.R. Secretory and viral fusion may share mechanistic events with fusion between curved lipid bilayers. Proc Natl Acad Sci USA. 95:1998;9274-9279.
50. 2+ sensitivity. J Cell Biol. 143:1998;1845-1857.
51. Tahara M., Coorssen J.R., Timmers K., Blank P.S., Whalley T., Scheller R.H., Zimmerberg J. Calcium can disrupt the SNARE protein complex on sea urchin egg secretory vesicles without irreversibly blocking fusion. J Biol Chem. 273:1998;33667-33673.
52. Ungermann C., Sato K., Wickner W. Defining the function of trans-SNARE pairs. Nature. 396:1998;543-548.
53. Yang B., Gonzalez L.C. Jr., Prekeris R., Steegmaier M., Advani R.J., Scheller R.H. SNARE interactions are not selective. Implications for membrane fusion specificity. J Biol Chem. 274:1999;5649-5653. Potential SNARE complexes are tested using five proteins of the vesicle-associated membrane protein (VAMP) family, three members of the SNAP-25 family, and three members of the syntaxin family. All 21 complexes have very similar biophysical properties, suggesting that specificity of membrane fusion is not encoded by the interactions between SNAREs.
54. Fasshauer D., Antonin W., Margittai M., Pabst S., Jahn R. Mixed and non-cognate SNARE complexes. Characterization of assembly and biophysical properites. J Biol Chem. 28:1999;15440-15446. Artificial SNARE complexes are tested using combinations of the SNAREs syntaxin 2, 3, 4, synaptobrevin, and endobrevin. The resulting complexes have very similar biophysical properties to the synaptic fusion complex of synaptobrevin-syntaxin-SNAP-25. This suggests that specificity of membrane fusion is probably not due to specific SNARE pairing.
55. Fischer von Mollard G., Nothwehr S.F., Stevens T.H. The yeast v-SNARE Vti1p mediates two vesicle transport pathways through interactions with the t-SNAREs Sed5p and Pep12p. J Cell Biol. 137:1997;1511-1524.
56. ••], where interactions between Sec1 and the assembled yeast SNARE complex are found.
57. Carr C.M., Grote E., Munson M., Hughson F.M., Novick P.J. Sec1p binds to SNARE complexes and concentrates at sites of secretion. J Cell Biol. 146:1999;333-344. Experiments in yeast show an interaction between Sec1p and the assembled yeast SNARE complex.
58. 2+-dependent and -independent activities of neural and non-neural synaptotagmins. Nature. 375:1995;594-599.
59. Schiavo G., Gmachl M.J., Stenbeck G., Söllner T.H., Rothman J.E. A possible docking and fusion particle for synaptic transmission. Nature. 378:1995;733-736.
60. Schiavo G., Stenbeck G., Rothman J.E., Söllner T.H. Binding of the synaptic vesicle v-SNARE, synaptotagmin, to the plasma membrane t-SNARE, SNAP-25, can explain docked vesicles at neurotoxin-treated synapses. Proc Natl Acad Sci USA. 94:1997;997-1001.
61. Chapman E.R., Desai R.C., Davis A.F., Tornehl C.K. Delineation of the oligomerization, AP-2 binding, and synprint binding region of the C2B domain of synaptotagmin. J Biol Chem. 273:1998;32966-32972.
62. 2+-independent interaction between the C2A-C2B domains and the synaptic SNARE complex is observed by native gel electrophoresis. A model of this super-complex is proposed.
63. 2+ binding loop of synaptotagmin with lipid bilayers. J Biol Chem. 273:1998;13995-14001.
64. Chae Y.K., Abildgaard F., Chapman E.R., Markley J.L. Lipid binding ridge on loops 2 and 3 of the C2A domain of synaptotagmin I as revealed by NMR spectroscopy. J Biol Chem. 273:1998;25659-25663.
65. 2+ regulates the interaction between synaptotagmin and syntaxin 1. J Biol Chem. 270:1995;23667-23671.
66. 2+ induce a conformational change? Biochemistry. 17:1998;16106-16115.
67. Stahl B., Chou J.H., Li C., Südhof T.C., Jahn R. Rab3 reversibly recruits rabphilin to synaptic vesicles by a mechanism analogous to raf recruitment by ras. EMBO J. 15:1996;1799-1809.
68. Orita S., Sasaki T., Naito A., Komuro R., Ohtsuka T., Maeda M., Suzuki H., Igarashi H., Takai Y. Doc2: a novel brain protein having two repeated C2-like domains. Biochem Biophys Res Commun. 206:1995;439-448.
69. Ubach J., Garcia J., Nittler M.P., Südhof T.C., Rizo J. Structure of the janus-faced C2B domain of rabphilin. Nat Cell Biol. 1:1999;106-112. The structure of the C2B domain of rabphilin is determined by NMR. It is structurally very similar to the C2B domain of synaptotagmin III [36].
70. 2+ concentration increase.
71. Søgaard M., Tani K., Ye R.R., Geromanos S., Tempst P., Kirchhausen T., Rothman J.E., Söllner T. A Rab protein is required for the assembly of SNARE complexes in the docking of transport vesicles. Cell. 78:1994;937-948.
72. Simons K., Zerial M. Rab proteins and the road maps for intracellular transport. Neuron. 11:1993;789-799.
73. Pfeffer S.R. Rab GTPases: master regulators of membrane trafficking. Curr Opin Cell Biol. 6:1994;522-526.
74. Geppert M., Südhof T.C. Rab3 and synaptotagmin: the yin and yang of synaptic membrane fusion. Annu Rev Neurosci. 21:1998;75-95.
75. Fischer von Mollard G., Mignery G.A., Baumert M., Perin M.S., Hanson T.J., Burger P.M., Jahn R., Südhofm T.C. Rab3A is a small GTP-binding protein exclusively localized to synaptic vesicles. Proc Natl Acad Sci USA. 87:1990;1988-1992.
76. Brennwald P., Novick P. Interactions of three domains distinguishing the Ras-related GTP-binding proteins Ypt1 and Sec4. Nature. 362:1993;560-563.
77. Dunn B., Stearns T., Botstein D. Specificity domains distinguish the Ras-related GTPases Ypt1 and Sec4. Nature. 362:1993;563-565.
78. Castillo P.E., Janz R., Südhof T.C., Tzounopoulos T., Malanka R.C., Nicoll R.A. Rab3A is essential for mossy fiber long-term potentiation in the hippocampus. Nature. 388:1997;590-593.
79. Wang Y., Okamoto M., Schmitz F., Hofmann K., Südhof T.C. Rim is a putative Rab3 effector in regulating synaptic-vesicle fusion. Nature. 388:1997;593-598.
80. Ostermeier C., Brunger A.T. Structural basis of Rab effector specificity: crystal structure of the small G protein Rab3A complexed with the effector domain of rabphilin-3A. Cell. 96:1999;363-374. The crystal structure of the Rab3A-rabphilin-3A complex is presented. Two contact regions are revealed. One of them involves the conserved switch I and switch II regions. The other one involves the hyper-variable carboxy-terminal region of Rab3A. It is speculated that the latter surface contact area could confer specificity for Rab-Rab-effector pairing.
81. Dumas J.J., Zhu Z., Connolly J.L., Lambright D.G. Structural basis of activation and GTP hydrolysis in Rab proteins. Structure. 7:1999;413-423. The structure of Rab3A bound to GppNHp (guanylyl-5′-[β,α-imido]-triphosphate) is presented.
82. ••].
83. Schalk I., Zeng K., Wu S-K., Stura E.A., Matteson J., Huang M., Tandon A., Wilson I.A., Balch W.E. Structure and mutational analysis of rab GDP-dissocation inhibitor. Nature. 381:1996;42-48.
84. Nischimune A., Isaac J.T., Molnar E., Noel J., Nash S.R., Tagaya M., Collingridge G.L., Henley J.M. NSF binding to GluR2 regulates synaptic transmission. Neuron. 21:1998;87-97.
85. Luscher C., Xia H., Beattie E.C., Carroll R.C., von Zastrow M., Malenka R.C., Nicoll R.A. Role of AMPA receptor cycling in synaptic transmission and plasticity. Neuron. 24:1999;649-658. AMPA receptor cycling is suggested to play a role in neuronal plasticity. A possible role of NSF is proposed in the exocytosis step of receptor cycling. Exocytosis of AMPA-receptor-containing vesicles would increase the density of receptors in the postsynaptic membrane, hence enhancing neurotransmission.
86. Fleming K.G., Hohl T.M., Yu R., Müller S.A., Wolpensinger B., Engel A., Engelhardt H., Brunger A.B., Söllner T.H., Hanson P.I. A revised model for the oligomeric state of the N-ethylmaleimide sensitive factor, NSF. J Biol Chem. 273:1998;15675-15681.
87. Beyer A. Sequence analysis of the AAA protein family. Protein Sci. 6:1997;2043-2058.
88. Sumida M., Hong R-M., Tayaga M. Role of two nucleotide-binding regions in an N-ethylmaleimide-sensitive factor involved in vesicle-mediated protein transport. J Biol Chem. 269:1994;20636-20641.
89. Hohl T.M., Paralti F., Wimmer C., Rothman J.E., Söllner T.H., Engelhardt H. Arrangement of subunits in 20S particles consisting of NSF, SNAPs, and SNARE complexes. Mol Cell. 2:1998;539-548.
90. Yu R.C., Hanson P.I., Jahn R., Brunger A.T. Structure of the ATP-dependent oligomerization domain of N-ethylmaleimide sensitive factor complexed with ATP. Nat Struct Biol. 5:1998;803. -811.
91. Lenzen C.U., Steinmann D., Whiteheart S.W., Weis W.I. Crystal structure of the hexamerization domain of N-ethylmaleimide-sensitive fusion protein. Cell. 94:1998;525-536.
92. Nagiec E.E., Bernstein A., Whiteheart S.W. Each domain of N-ethylmaleimide-sensitive fusion protein contributes to its transport activity. J Biol Chem. 49:1995;29182-29188.
93. ••].
94. May A.P., Misura K.M., Whiteheart S.W., Weis W.I. Crystal structure of the amino-terminal domain of N-ethylmaleimide-sensitive fusion protein. Nat Cell Biol. 1:1999;175-182. An independent crystal structure of NSF-N is presented that contains three molecules per asymmetric unit. It is speculated that the trimeric packing could resemble the oligomeric arrangement of NSF-N, with three NSF-N domains pointing inwards and three pointing outwards.
95. Babor S.M., Fass D. Crystal structure of the Sec18P N-terminal domain. Proc Natl Acad Sci USA. 96:1999;4759-14764. The crystal structure of the Sec18p amino-terminal domain is presented. It is very similar to the amino-terminal domain of NSF (NSF-N).
96. Coles M., Diercks T., Liermann J., Groger A., Rockel B., Baumeister W., Koretke K.K., Lupas A., Peters J., Kessler H. The solution structure of VAT-N reveals a 'missing link' in the evolution of complex enzymes from a simple βαββ element. Curr Biol. 9:1999;1158-1168. The solution structure of VAT-N demonstrates a structural similarity between the amino-terminal domains of the archaebacterium Thermoplasma acidophilum and NSF.
97. Rice L.M., Brunger A.T. Crystal structure of the vesicular transport protein Sec17: implications for SNAP function in SNARE complex disassembly. Mol Cell. 4:1999;85-95. The crystal structure of Sec17, the yeast homologue of α-SNAP, is presented. The structure consists of a twisted sheet consisting of α-helical hairpins and a globular carboxy-terminal domain. The twisted sheet probably interacts with the SNARE complex while the carboxyl terminus interacts with the amino-terminal domain of NSF.
98. Das A.K., Cohen P.T.W., Barford D. The structure of the tetratricopeptide repeats of protein phosphatase 5: implications for TPR-mediated protein-protein interactions. EMBO J. 17:1998;1192-1199.
99. Yaffe M.B., Rittinger K., Volinia S., Caron P.R., Aitken A., Leffers H., Gamblin S.J., Smerdon S.J., Cantley L.C. The structural basis for 14-3-3:phosphopeptide binding specificity. Cell. 91:1997;961-971.
100. Groves M.R., Hanlon N., Turowski P., Hemmings B.A., Barford D. The structure of the protein phosphatase 2A PR65/A subunit reveals the conformation of its 15 tandemly repeated HEAT motifs. Cell. 96:1999;99-110. The structure of protein phosphatase 2A PR65/A subunit is presented. This protein contains 15 HEAT repeats.
101. Ybe J.A., Brodsky F.M., Hofmann K., Lin K., Liu S-H., Chen L., Earnest T.N., Fletterick R., Hwang P.K. Clathrin self-assembly is mediated by a tandemly repeated superhelix. Nature. 399:1999;371-395. The structure is presented of a clathrin fragment that is involved in mediating spontaneous clathrin heavy-chain polymerization and light-chain association. This clathrin fragment has structural similarity to HEAT and TRP repeats, and Sec17.
102. Kee Y., Lin R.C., Hsu S-C., Scheller R.H. Distinct domains of syntaxin are required for synaptic vesicle fusion complex formation and dissociation. Neuron. 14:1995;991-998.
103. Hanson P.I., Otto H., Barton N., Jahn R. The N-ethylmaleimide-sensitive fusion protein and α-SNAP induce a conformational change in syntaxin. J Biol Chem. 270:1995;16955-16961.
104. Hayashi T., Yamasaki S., Nauenburg S., Binz T., Niemann H. Disassembly of the reconstituted synaptic vesicle membrane fusion complex in vitro. EMBO J. 14:1995;2317-2325.
105. Wei S., Xu T., Ashery U., Kollewe A., Matti U., Antonin W., Rettig J., Neher E. Exocytotic mechanism studied by truncated and zero layer mutants of the C-terminus of SNAP-25. EMBO J. 19:2000;1279-1289. In order to study the role of the ionic layer in the SNARE complex, the authors used the Semliki Forest virus (SFV) system to infect adrenal chromaffin cells with SNAP-25 Q174L, a point mutation of the ionic layer. Cells expressing the mutant displayed a selective reduction in the sustained phase of exocytosis, whereas the two components of the exocytotic burst remained unaffected. Furthermore, the exocytotic response to the second flash was significantly reduced, indicating a defect in refilling kinetics. It was concluded that the ionic layer is critical for the formation of SNARE complexes. It is conceivable that it is also required for disassembly and subsequent endocytosis.