©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Biochemistry of Neurotransmitter Secretion(*)

Sandra M. Bajjalieh Richard H. Scheller

From the Howard Hughes Medical Institute, Department of Molecular and Cellular Physiology, Stanford University, Stanford, California 94305-5426

INTRODUCTION
Neurosecretion Utilizes Mechanisms Common to All Eukaryotic Membrane Transport
Calcium Regulation Is Superimposed on the Machinery of Membrane Trafficking
Summary
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


INTRODUCTION

The release of neurotransmitter via regulated exocytosis is the primary mode of communication in the nervous system. Changes in the efficacy of this process are at the basis of nervous system plasticity. Our understanding of the biochemical events that mediate neurotransmitter secretion has undergone a revolution in the last few years. The initial focus of research, identifying proteins associated with transmitter-containing (synaptic) vesicles, has spawned the current functional analyses of synapse proteins. These analyses have revealed that neurotransmitter release utilizes molecular events common to all membrane trafficking.

Neurotransmitters are released from small (50 nM) vesicles that undergo many rounds of fusion and recycling at presynaptic terminals. Synaptic vesicles appear to be assembled not in the Golgi but rather in presynaptic endosomal compartments(1, 2) . Unlike neuropeptide-containing secretory granules, synaptic vesicles fill with transmitter after they are formed. Filling with neurotransmitter is mediated by specific transport molecules(3) . Loaded vesicles either sequestered in a reserve pool via interaction with cytoskeletal elements (4) or cluster at the presynaptic terminal at specialized regions termed active zones. The fusion of these ``docked'' vesicles with the presynaptic plasma membrane occurs when intracellular calcium concentrations rise during an action potential. Calcium regulates one of the final events in synaptic vesicle fusion because the release of neurotransmitter following calcium elevation occurs in less than 1 ms.

Neurotransmitter release is therefore the sum of many molecular processes. This minireview will concentrate on what has been learned about vesicle targeting and fusion from a combination of yeast genetics and biochemical analyses. How calcium might regulate these events is discussed in light of this common pathway.


Neurosecretion Utilizes Mechanisms Common to All Eukaryotic Membrane Transport

The discovery that several proteins localized to the synapse are homologs of yeast proteins required for constitutive secretion provided the first solid evidence that neurosecretion is a specialized version of general membrane trafficking(5) . This realization has led to the search for non-synaptic forms of these proteins in animal cells and for mammalian homologs of other secretory yeast proteins(6) . While neurosecretion is distinguished from constitutive secretion by being regulated by calcium, it shares features with all membrane trafficking including the mechanisms that mediate vesicle targeting and docking and the involvement of the actin cytoskeleton and acidic phospholipids.

Docking and Fusion of Vesicles at the Presynaptic Membrane Are Proposed to Occur through a Series of Protein-Protein Interactions

Like all vesicles that mediate transport from one membrane compartment to another, synaptic vesicles must find the appropriate acceptor membrane. Targeting would be most easily accomplished if vesicles contained proteins that specifically interact with molecules localized to the acceptor membrane. Thus the hypothesis, recently named the SNARE (^1)hypothesis (7) , states that each class of transport vesicle contains a specific targeting protein (v- or vesicle SNARE) that is capable of associating only with a receptor protein (t- or target SNARE) specific to the appropriate acceptor membrane. The proteins hypothesized to serve as SNAREs at the synapse include the synaptic vesicle protein VAMP (vesicle-associated membrane protein, also called synaptobrevin)(8, 9) and two plasma membrane proteins, syntaxin (also HPC-1) (10, 11) and SNAP-25 (synapse-associated protein of 25 kDa)(12) . To identify the series of molecular events that mediate exocytosis, protein cross-linking studies and immunoprecipitation experiments are being combined with the genetic dissection of secretion in yeast. These studies have produced the working model illustrated in Fig. 1.


Figure 1: A series of protein interactions hypothesized to mediate vesicle docking and fusion. The current working model of vesicle docking and fusion proposes that specificity of vesicle docking is provided by the specific interactions of the proteins VAMP, syntaxin, and SNAP-25. See text for a detailed description.



Before vesicles dock, the v-SNARE VAMP is hypothesized to be associated with the synaptic vesicle protein synaptophysin. On the plasma membrane, the t-SNARE syntaxin is bound to a soluble protein called n-Sec1. Syntaxin and n-Sec1 dissociate by an unknown mechanism (i) allowing the formation of a protein complex that sediments at 7 S. This complex includes VAMP, syntaxin, the t-SNARE SNAP-25, and the synaptic vesicle protein synaptotagmin. Since synaptotagmin is expressed only in neural and endocrine cells, the 7 S complex may be unique to regulated secretion. Synaptotagmin dissociates from the 7 S complex (ii) allowing the addition of the soluble protein alpha-SNAP (soluble NSF attachment protein). As its name suggests, the binding of alpha-SNAP allows the subsequent binding of NSF (N-ethylmaleimide-sensitive fusion protein) and formation of a 20 S complex (iii). NSF is an ATPase, and its activity (iv) results in the disruption of the 20 S complex. The dissociation of the 20 S particle may initiate membrane fusion via unknown intermediates as illustrated in step v.

Evidence for this model includes the protein interactions discussed below and the following observations. 1) VAMP and syntaxin each have homologs in yeast (5) and in animal cells(13, 14) . In yeast, unique homologs of these proteins exist at different stages of the secretory pathway where they are required for secretory vesicle docking and/or fusion. 2) VAMP, syntaxin, and SNAP-25 are substrates of the clostridial and tetanus neurotoxins that block neurosecretion, an observation that indicates that these proteins are involved in the exocytosis of synaptic vesicles(15) .

The VAMP-Synaptophysin Complex

In situ cross-linking of synaptic proteins has revealed that VAMP is associated with the synaptic vesicle protein synaptophysin(16, 17) . Synaptophysin is specific to small clear secretory vesicles and forms homomultimeric complexes that exhibit a calcium-dependent ion conductance when reconstituted into lipid bilayers(18) . It is not known precisely where the VAMP-synaptophysin interaction is positioned in the sequence of events. Since synaptophysin is not part of either the 7 or 20 S complexes, its interaction with VAMP is predicted to precede them.

The Syntaxin-n-Sec1 Complex

Syntaxin forms a complex with the soluble protein n-Sec1, which is named for its homology to the yeast Sec1 protein(19, 20) . This complex can be immunoprecipitated from solubilized vesicles and can also be formed in vitro between recombinant proteins. Several Sec1 homologs are present in yeast, each specific to a single step in the secretory pathway(21) . The syntaxin-n-Sec1 interaction displays the highest affinity of all syntaxin interactions characterized (K(d) = 80 nM)(20) . This complex is hypothesized to form prior to other syntaxin interactions because overexpression of yeast syntaxin homologs suppresses mutations in the Sec1 protein(22) . Therefore, n-Sec1 may act as either a chaperone, inducing the correct conformation for syntaxin interaction with other SNAREs, or as a regulator of syntaxin availability (or both).

The 7 S Complex

The 7 S complex has been isolated from detergent-solubilized synaptosomes(23) . It is hypothesized to initiate the docking of vesicles at the presynaptic membrane through specific interactions between VAMP, syntaxin, and SNAP-25. This hypothesis is supported by binding studies utilizing recombinant proteins. For example, the synaptic vesicle-specific VAMP isoforms 1 and 2 bind neural specific syntaxin isoforms 1A and 1B and syntaxin 4 but not the non-neural syntaxins 2 and 3(24) . These interactions, while specific, are not of high affinity; the apparent K(d) values are in the micromolar range. However, the affinity of VAMP-syntaxin interactions is much higher in the presence of SNAP-25. This effect of SNAP-25 is also isoform-specific. SNAP-25 facilitates VAMP 2-syntaxin 1 binding but does not significantly affect the binding of VAMP 2 to syntaxin 4(20) , indicating that additional targeting specificity is provided by three-way protein interactions. This specificity in the interactions of VAMP, syntaxin, and SNAP-25 supports the hypothesis that they mediate targeting of synaptic vesicles to the presynaptic membrane. However, the specificity of targeting may not be as simple as the binding of a single v-SNARE to t-SNARE. Immunoprecipitation of the yeast t-SNARE Sed5p co-precipitates several proteins that may function as v- or t-SNAREs, indicating that targeting specificity may be coded for by combinations of vesicle and target membrane proteins.

The 20 S Complex

The 7 S complex is converted to a 20 S complex with the addition of alpha-SNAP and subsequent binding of NSF(23) . alpha-SNAP and NSF are the mammalian homologs of two proteins required for secretion in yeast, Sec18p and Sec17p(25) . Unlike the other proteins with yeast homologs described so far, NSF and alpha-SNAP are not specific to a given transport step but rather act at most stages of the secretory pathway. In fact, they were first identified as soluble proteins required for ER to Golgi transport in animal cells (26) . In addition to being formed in vitro, the 20 S complex can be isolated from detergent-solubilized synaptosomes(20) . It is isolated in the presence of non-hydrolyzable forms of ATP or magnesium chelators, and addition of Mg/ATP disrupts the complex, presumably due to the ATPase activity of NSF. Disruption of the 20 S complex may initiate fusion. This hypothesis suggests that targeting specificity could occur with fusion, rather than with docking, and is supported by the observation that vesicles remain docked but do not fuse in tetanus toxin-treated neurons(27) .

Small GTP-binding Proteins Are Required for Vesicle Targeting and Docking in Yeast and May Mediate a Similar Function in Animal Cells

Genetic analyses of secretion in yeast implicate small Ras-like GTP-binding proteins in membrane trafficking. As is the case with the SNAREs, each step of the secretory pathway has an associated small GTP-binding protein. For example, the products of the YPT1 and SEC4 genes are required for ER to Golgi and Golgi to plasma membrane transport, respectively(28) . Ypt1p and Sec4p are associated with transport vesicles, and mutations in either result in an accumulation of vesicles(29, 30, 31) . These observations suggest that these proteins participate in vesicle targeting. Their action is thought to occur prior to the formation of the 20 S SNARE complex for two reasons: 1) mutations in Ypt1 and Sec4 can be suppressed by overexpression of the components of the SNARE complex (22) and 2) the 20 S complex does not form in ypt1 mutants (32) . A potential role for small GTP-binding proteins is to prime or activate members of the targeting complex, although no direct interaction between small GTP-binding proteins and SNARE proteins has been reported.

Small GTP-binding proteins are also associated with transport vesicles in animal cells(33) . As in yeast, each species of transport vesicle contains a specific isoform. Rab3, the isoform specific to synaptic vesicles, has been hypothesized to play a role identical to that of Sec4p in yeast. However, mutations that produce a dominant negative phenotype in yeast do not disrupt secretion in animal cells(34) , and targeted disruption of the rab3 gene in mice produces only a mild phenotype(35) . These results may indicate that Rab3 performs a redundant function in neurons. However, they may also indicate either that Rab3 is not the mammalian functional homolog of Sec4p and Ypt1p or that its function is less important in the synapse than in constitutive secretion.

Acidic Phospholipids Are Required for Both Constitutive and Regulated Secretion

The identification of a yeast protein required for secretion as a phosphatidylinositol (PI) transfer protein (36) provided the first indication that secretion relies on dynamic regulation of membrane lipid content. This protein, Sec14p, exchanges PI for phosphatidylcholine in vitro and is hypothesized to regulate the PI content of membranes. Suppressors of the sec14 mutant phenotype affect the phosphatidylcholine synthesis pathway (37, 38) suggesting that the ratio of phosphatidylcholine to PI in membranes is important for normal secretion. A role for acidic phospholipids in regulated secretion has emerged from characterization of soluble factors required to reconstitute regulated secretion in disrupted neuroendocrine cells. Two proteins that mediate the ATP requirement in this system are the PI transfer protein and a PIP kinase, indicating that PI and its phosphorylated forms, PIP and PIP(2), participate in regulated secretion(39, 40) .

There are several ways acidic phospholipids could play important roles in membrane transport. Acidic phospholipids may act as fusogens(41, 42) . This hypothesis is based on the observation that an increase in the acidic phospholipid content of synthetic liposomes increases their fusability(43) . Alternatively, acidic phospholipids may regulate the interaction of cytoskeletal proteins with vesicles. PI and its higher phosphorylated forms, PIP and PIP(2), regulate the activity of many actin-associated proteins(44) . Therefore, changes in the concentrations of these lipids in either the vesicle or acceptor membrane are predicted to alter the surrounding actin cytoskeleton. This potential link between membrane composition and actin regulatory proteins is especially appealing in light of observations that changes in the actin cytoskeleton accompany secretion.

The Actin Cytoskeleton Participates in Secretion

Both constitutive and regulated secretion involve dynamic changes in the actin cytoskeleton. Dissolution of actin filaments occurs with secretion(45) , and drugs that induce actin filament disassembly can induce exocytosis(46) . Treatment with reagents that depolymerize actin filaments is sufficient to trigger secretion in permeabilized exocrine cells. However, a minimal actin structure is required since treatment with high concentrations of these reagents abolishes secretion. In addition, antibodies directed against the actin-organizing protein fodrin inhibit regulated secretion in endocrine cells(47) . Taken together these observations indicate that a delicate regulation of the actin cytoskeleton is required for normal secretion.

What is the role of the cytoskeleton in effecting secretion? One clue has come from the identification of a protein required for vesicle docking that has homology to several actin-binding proteins. This protein, termed TAP (transcytosis-associated protein) (48) or p115 (78) was identified through the characterization of soluble proteins required for transcytotic transport in epithelial cells. TAP is required, along with NSF, for the docking and fusion of transport vesicles with the plasma membrane. TAP appears to act prior to NSF to mediate vesicle docking at the acceptor membrane since vesicles incubated with TAP in the absence of ATP will dock, but will not fuse with, the plasma membrane(48) . TAP is a homolog of the yeast USO1 gene product. Mutations in the USO1 gene disrupt ER to Golgi transport indicating that TAP and Uso1p may perform a similar function required at all stages of the secretory pathway(49) . Given its homology to actin-associated proteins and its role in vesicle docking and association with transport vesicles(50) , TAP may mediate cytoskeletal assisted docking of vesicles as illustrated in Fig. 2A. In this model, TAP links transport vesicles to actin filaments and presents the vesicle to the acceptor membrane. TAP may act alone or in conjunction with another myosin-like protein(s). Membrane docking and fusion are then achieved via specific v- and t-SNARE interaction.


Figure 2: Cytoskeleton-associated protein may act at both vesicle docking and fusion. A, TAP (p115) is required for docking of transport vesicles with the plasma membrane. TAP is associated with transport vesicles and contains a region homologous to actin-binding proteins. TAP may therefore facilitate the formation of the 20 S particle by positioning the vesicle over the t-SNARE complex. B, CAPS may resolve docked hemi-fused vesicles toward fusion. This hypothesis predicts that CAPS function would be mediated by a non-calcium-regulated homolog in constitutive secretion. CAPS may also act prior to the formation of the 20 S particle in a fashion similar to that hypothesized for TAP.




Calcium Regulation Is Superimposed on the Machinery of Membrane Trafficking

While it has become apparent that neurotransmitter secretion utilizes mechanisms common to all membrane transport, it is distinguished by its regulation by calcium. Calcium-regulated secretion is also a feature of endocrine and exocrine cells. Two to three calcium-mediated processes have been revealed by physiological analyses of regulated secretion in endocrine cells(51, 52, 53) . These processes, which differ in their rates and calcium affinity, have been hypothesized to reflect calcium-dependent vesicle priming and fusion (52) . Vesicle fusion requires higher calcium concentrations (K(d) = 27 µM) than priming, which can be mediated by less than 1 µM calcium. In neurons, the initiation of fusion begins approximately 10 times faster than in endocrine cells and requires even higher calcium concentrations (>100 µM calcium)(54) . Therefore, the calcium regulation of neural secretion may utilize the high and medium affinity calcium regulators present in endocrine cells (requiring submicromolar to micromolar calcium) as well as a low affinity regulator (requiring millimolar calcium).

Synaptotagmin May Function as a Calcium Sensor

The synaptic vesicle protein synaptotagmin contains two C2 domains common to proteins that exhibit calcium-induced membrane association(55, 56) . The presence of these domains led to the proposal that synaptotagmin performs one of the calcium-regulated events of regulated secretion. This proposal is supported by the observations that synaptotagmin binds lipids in a calcium-dependent fashion (57, 58, 59) and that calcium-dependent exocytosis is inhibited when anti-synaptotagmin antibodies or peptide fragments are injected into neuroendocrine cells (60) or neurons(61) . The presence of synaptotagmin in the 7 S complex suggests that it functions as an inhibitor of the constitutive secretory processes by preventing formation of the 20 S complex. This hypothesis predicts that the dissociation of synaptotagmin from the 7 S complex is regulated by calcium. While calcium-regulated dissociation of the 7 S particle has not been observed(23) , analyses performed thus far have been in the absence of phospholipids, which are required for synaptotagmin-calcium interaction.

Disruption of the synaptotagmin gene in Caenorhabditiselegans and Drosophila produces animals that are severely impaired but maintain limited levels of synaptic transmission, indicating that synaptotagmin is not absolutely required for neurotransmission(62, 63) . Likewise, disruption of the synaptotagmin I gene in mice produces animals that die within 2 days but which are morphologically indistinguishable from normal mice at birth(64) . Analyses of synaptic transmission in synaptotagmin-deficient animals have produced somewhat disparate results. In Drosophila, stimulus evoked neurotransmission at the neuromuscular junction is severely impaired in synaptotagmin null mutants(65, 66, 67) . However, spontaneous transmission at this synapse is increased severalfold. This increased spontaneous release would be predicted to deplete the vesicle pool and therefore diminish evoked responses. These results support the hypothesis that synaptotagmin acts as an inhibitor of constitutive secretion. The absence of synaptotagmin I also produces markedly attenuated synaptic transmission in mice. However, unlike Drosophila synaptotagmin null mutants, cultured hippocampal neurons from synaptotagmin-deficient mice do not display an increase in spontaneous transmitter release. The evoked transmission that remains appears to lack the large, early component while retaining a late, smaller component. These observations suggest that synaptotagmin could mediate the rapid, initial events of transmitter release and argue against it serving as a negative regulator of transmission(64) .

It is unlikely that synaptotagmin performs a different function in mammalian neurons than it does in the neurons of flies. Therefore, it is difficult to rationalize the differences in spontaneous transmitter release between synaptotagmin-deficient flies and mice. These observed differences could reflect inherent differences in the preparations studied (embryonic neuromuscular junction in flies versus cultured central nervous system neurons from mice). Each system may highlight different aspects of synaptotagmin functioning. Synaptotagmin may in fact perform several functions. It is associated with the clathrin adaptor protein AP2(69) , an interaction that implicates synaptotagmin in endocytosis. In addition, quantitative electron microscopic analysis of synapses indicates that synaptotagmin null flies have fewer synaptic vesicles immediately adjacent to the plasma membrane suggesting that synaptotagmin also plays a role in membrane docking(68) . Two observations suggest that synaptotagmin may not be the low affinity calcium regulator of fast neurotransmitter release. First, its affinity for calcium is in the micromolar range(58) , which is significantly higher than the millimolar calcium concentrations required to induce fast transmission(54) . Second, synaptotagmin is present in endocrine cells, which do not exhibit the fast transmission of neurons.

p145 CAPS May Mediate One of the Last Events of Exocytosis

Calcium-regulated secretion has been reconstituted in disrupted neuroendocrine cells and shown to require cytosol, Mg/ATP, and calcium(70) . This system is being used to identify soluble factors required for exocytosis and has led to the identification of proteins required for ATP- and calcium-dependent events(39, 71, 72) . Calcium-regulated exocytosis in these cells requires a soluble 145-kDa protein, termed CAPS (calcium-activated protein for secretion). CAPS performs a function that occurs after all ATP-dependent processes (71) and is therefore predicted to act after the dissolution of the 20 S complex. CAPS binds actin in a calcium-dependent fashion (73) and therefore could function either as a regulator of cytoskeletal assembly or disassembly or to regulate actin-mediated vesicle movement. Given its apparent late action, a possible action of CAPS is to provide mechanical energy to resolve hemi-fused membranes to an open fusion pore (Fig. 2B). In addition to this late action, movement of vesicles by CAPS-like proteins could also participate in the positioning of vesicles. CAPS and proteins such as TAP may therefore perform a function both prior to and after the interaction of docking proteins (Fig. 2). This function can be imagined as a conveyor belt-like movement of vesicles along actin filaments that both positions vesicles for docking and provides the required energy to resolve fusion. It should be pointed out, however, that although CAPS may mediate one of the last events of regulated secretion, it is also not likely to be the mediator of the very rapid events of neurosecretion. As with synaptotagmin, CAPS requires only micromolar calcium for activity and is also present in endocrine cells as well as in neurons.

Does Neurotransmitter Secretion Utilize a Unique Calcium-dependent Event?

Despite the similarities of neurotransmitter release to endocrine secretion, neurosecretion has the unique property of being more rapid (200-500 µs (74) versus ms in endocrine cells(52, 75) ) and requiring higher calcium concentrations (millimolar versus micromolar). It has been hypothesized that the difference in rate is mediated by the association of synaptic vesicles with calcium channels clustered in active zones of the synapse(73) . Both synaptotagmin and syntaxin have been reported to interact with the N-type calcium channel(10, 76) . In addition, another synaptic vesicle protein, cysteine string protein, was identified by its ability to regulate N-type calcium channel activity(77) , suggesting that vesicles contain a protein that regulates the influx of calcium in their vicinity.

Is there an additional calcium regulator present in neurons? Given the speed of the initial events of neurotransmitter release, it is possible that a protein does not mediate this low affinity calcium-regulated step. Rapid fusion may instead be mediated by the presence of acidic phospholipids, which are fusogenic in the presence of millimolar calcium concentrations(43) . This hypothesis proposes that, in addition to the other calcium-regulated events, neurons, by positioning vesicles near calcium channels, utilize the low calcium affinity fusogenic properties of acidic phospholipids to produce the fast component of neural secretion.


Summary

The progress that has resulted from the convergence of biochemistry with yeast genetics has accelerated the pace at which the molecular events of membrane transport are being elucidated. Future research will focus not only on testing the proposed sequence of protein-protein interactions but also on identifying how calcium regulation is imposed on this system. As our understanding of the basic mechanisms of neurosecretion increases, attention will undoubtedly shift to how the molecules of release are modified to produce changes in synaptic efficacy.


FOOTNOTES

*
This minireview will be reprinted in the 1995 Minireview Compendium, which will be available in December, 1995.

(^1)
The abbreviations used are: SNARE, soluble NSF attachment protein receptor; v-SNARE, vesicle SNARE; t-SNARE, target SNARE; VAMP, vesicle-associated membrane protein; SNAP-25, synapse-associated protein of 25 kDa; NSF, N-ethylmaleimide-sensitive fusion protein; ER, endoplasmic reticulum; PI, phosphatidylinositol; PIP, phosphatidylinositol phosphate; TAP, transcytosis-associated protein; CAPS, calcium-activated protein for secretion.


ACKNOWLEDGEMENTS

We thank Tom Martin, Jesse Hay, and Tom Schwarz for helpful discussions and John Guastella and Ludolf von Rüden for critical review of the manuscript.


REFERENCES

  1. McPherson, P. S., and DeCamilli, P. (1994) Semin. Neurosci. 6, 137-148 [CrossRef]
  2. Huttner, W. B., and Tooze, S. A. (1989) Curr. Opin. Cell Biol. 1, 648-654 [Medline] [Order article via Infotrieve]
  3. Edwards, R. H. (1992) Curr. Opin. Neurobiol. 2, 586-594 [Medline] [Order article via Infotrieve]
  4. Greengard Valtorta, P., Czernik, F., and Benfenati, A. J. F. (1993) Science 259, 780-785 [Medline] [Order article via Infotrieve]
  5. Bennett, M. K., and Scheller, R. H. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2559-2563 [Abstract]
  6. Shekman, R. (1992) Curr. Opin. Cell Biol. 4, 587-592 [Medline] [Order article via Infotrieve]
  7. Söllner, T., Whiteheart, S. W., Brunner, M., Erdjument-Bromage, H., Geromanos, S., Tempst, P., and Rothman, J. E. (1993) Nature 362, 318-324 [CrossRef][Medline] [Order article via Infotrieve]
  8. Trimble, W. S., Cowan, D. M., and Scheller, R. H. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 4538-4542 [Abstract]
  9. Baumert, M., Maycox, P. R., Navone, F., De Camilli, P., and Jahn, R. (1989) EMBO J. 8, 379-384 [Abstract]
  10. Bennett, M. K., Calakos, N., and Scheller, R. H. (1992) Science 257, 255-259 [Medline] [Order article via Infotrieve]
  11. Inoue, A., Obata, K., and Akagawa, K. (1992) J. Biol. Chem. 267, 10613-10619 [Abstract/Free Full Text]
  12. Oyler, G. A., Higgins, G. A., Hart, R. A., Battenberg, E., Billingsley, M., Bloom, F. E., and Wilson, M. C. (1989) J. Cell Biol. 109, 3039-3052 [Abstract]
  13. McMahon, H. T., Ushkaryov, Y. A., Edelmann, L., Link, E., Binz, T., Niemann, H., Jahn, R., and Sudhof, T. C. (1993) Nature 364, 346-349 [CrossRef][Medline] [Order article via Infotrieve]
  14. Bennett, M., Garcia-Arraras, J. E., Elferink, L., Peterson, K., Fleming, A. M., Hazuka, C. D., and Scheller, R. H. (1993) Cell 74, in press
  15. Montecucco, C., and Schiavo, G. (1993) Trends Biochem. Sci. 18, 324-327 [CrossRef][Medline] [Order article via Infotrieve]
  16. Calakos, N., and Scheller, R. H. (1994) J. Biol. Chem. 269, 24534-24537 [Abstract/Free Full Text]
  17. Edelmann, L., Chapman, E., and Jahn, R. (1994) Soc. Neurosci. Abstr. 20, 146.10
  18. Thomas, L., Hartung, K., Langosssch, D., Rehm, H., Bamberg, E., Franke, W. W., and Betz, H. (1988) Science 242, 1050-1053 [Medline] [Order article via Infotrieve]
  19. Pevsner, J., Hsu, S.-C., and Scheller, R. H. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 1445-1449 [Abstract]
  20. Pevsner, J., Hsu, S.-C., Braun, J. E., Calakos, N., Ting, A. E., Bennett, M. K., and Scheller, R. H. (1994) Neuron 13, 353-361 [Medline] [Order article via Infotrieve]
  21. Aalto, M. K., Keränen, S., and Ronne, H. (1992) Cell 68,
  22. Aalto, M. K., Ronne, H., and Keränen, S. (1993) EMBO J. 12, 4095-4104 [Abstract]
  23. Söllner, T., Bennett, M. K., Whiteheart, S. W., Scheller, R. H., and Rothman, J. E. (1993) Cell 75, 409-419 [Medline] [Order article via Infotrieve]
  24. Calakos, N., Bennett, M. K., and Peterson, K. E. (1994) Science 263, 1146-1149 [Medline] [Order article via Infotrieve]
  25. Kaiser, C. A., and Schekman, R. (1990) Cell 61, 723-733 [Medline] [Order article via Infotrieve]
  26. Malhotra, V., Orci, L., Glick, B. S., Block, M. R., and Rothman, J. E. (1988) Cell 54, 221-227 [Medline] [Order article via Infotrieve]
  27. Hunt, J. M., Bommert, K., Charlton, M. P., Kistner, A., Habermann, E., Augustine, G. J., and Betz, H. (1994) Neuron 1269-1279
  28. Ferro-Novick, S., and Novick, P. (1993) Annu. Rev. Cell Biol. 9, 575-599 [CrossRef]
  29. Segev, N., Mulholland, J., and Botstein, D. (1988) Cell 52, 915-924 [Medline] [Order article via Infotrieve]
  30. Ferro-Novick, S., Newman, A. P., Groesch, M., Ruohola, H., Rossi, G., Graf, J., and Shim, J. (1991) Cell Biophys. 19, 25-33 [Medline] [Order article via Infotrieve]
  31. Segev, N. (1991) Science 252, 1553-1556 [Medline] [Order article via Infotrieve]
  32. Sogaard, M., Tani, K., Ye, R. R., Geromanos, S., Tempst, P., Kirchhausen, T., Rothman, J. E., and Söllner, T. (1994) Cell 78, 937-948 [Medline] [Order article via Infotrieve]
  33. Zerial, M., and Stenmark, H. (1993) Curr. Opin. Cell Biol. 5, 613-620 [Medline] [Order article via Infotrieve]
  34. Ngsee, J. K., Fleming, A. M., and Scheller, R. H. (1993) Mol. Biol. Cell 4, 747-756 [Abstract]
  35. Geppert, M., Bolshakov, V. Y., Siegelbaum, S. A., Takel, K., De Camilli, P., Hammer, R. E., and Südof, T. C. (1994) Nature 369, 493-497 [CrossRef][Medline] [Order article via Infotrieve]
  36. Bankaitis, V. A., Aitken, J. R., Cleves, A. E., and Dowhan, W. (1990) Nature 347, 561-562 [CrossRef][Medline] [Order article via Infotrieve]
  37. Cleves, A. E., Novick, P. J., and Bankaitis, V. A. (1989) J. Cell Biol. 109, 2939-2950 [Abstract]
  38. Cleves, A. E., and Bankaitis, V. A. (1992) Adv. Microb. Physiol. 33, 73-144 [Medline] [Order article via Infotrieve]
  39. Hay, J. C., and Martin, T. F. J. (1993) Nature 366, 572-575 [CrossRef][Medline] [Order article via Infotrieve]
  40. Hay, J. C. (1994) Ph.D. dissertation, University of Wisconsin
  41. Shinghal, R., Scheller, R. H., and Bajjalieh, S. M. (1993) J. Neurochem. 61, 2279-2285 [Medline] [Order article via Infotrieve]
  42. Liscovitch, M., Chalifa, V., Pertile, P., Chen, C.-S., and Cantley, L. C. (1994) J. Biol. Chem. 269, 21403-21406 [Abstract/Free Full Text]
  43. Koter, M., De Kruijff, B., and Van Deenen, L. L. M. (1978) Biochim. Biophys. Acta 514, 255-263 [Medline] [Order article via Infotrieve]
  44. Janmey, P. A. (1994) Annu. Rev. Physiol. 56, 169-191 [CrossRef][Medline] [Order article via Infotrieve]
  45. Koffer, A., Tatham, P. E. R., and Gomperts, P. D. (1990) J. Cell Biol. 111, 919-927 [Abstract]
  46. Muallem, S., Kwiatkowska, K., Xu, X., and Yin, H. L. (1994) J. Cell Biol. , in press
  47. Perrin, D., Langley, O. K., and Aunis, D. (1987) Nature 326, 498-501 [CrossRef][Medline] [Order article via Infotrieve]
  48. Barroso, M. R., Nelson, D. S., and Sztul, E. S. (1994) Proc. Natl. Acad. Sci. U. S. A. , in press
  49. Nakajima, H., Hirata, A., Ogawa, A., Yonehara, T., Yoda, K., and Yamasaki, M. (1991) J. Cell Biol. 113, 245-260 [Abstract]
  50. Sztul, E., Kaplin, A., Saucan, L., and Palade, G. (1991) Cell 64, 81-89 [Medline] [Order article via Infotrieve]
  51. Neher, E., and Zucker, R. S. (1993) Neuron 10, 21-30 [Medline] [Order article via Infotrieve]
  52. Thomas, P., Wong, J. G., Lee, A. K., and Almers, W. (1993) Neuron 11, 93-104 [Medline] [Order article via Infotrieve]
  53. von Rüden, L., and Neher, E. (1993) Science 262, 1061-1065 [Medline] [Order article via Infotrieve]
  54. Heidelberger, R., Heinemann, C., Neher, E., and Matthews, G. (1994) Nature 371, 513-515 [CrossRef][Medline] [Order article via Infotrieve]
  55. Perin, M. S., Fried, V. A., Mignery, G. A., Jahn, R., and Sudhof, T. C. (1990) Nature 345, 260-263 [CrossRef][Medline] [Order article via Infotrieve]
  56. Wendland, B., Miller, K. G., Schilling, J., and Scheller, R. H. (1991) Neuron 6, 993-1007 [Medline] [Order article via Infotrieve]
  57. Brose, N., Petrenko, A. G., Sudhof, T. C., and Jahn, R. (1992) Science 256, 1021-1025 [Medline] [Order article via Infotrieve]
  58. Davletov, B. A., and Südhof, T. C. (1993) J. Biol. Chem. 268, 26386-26390 [Abstract/Free Full Text]
  59. Chapman, E. R., and Jahn, R. (1994) J. Biol. Chem. 269, 5735-5741 [Abstract/Free Full Text]
  60. Elferink, L. A., Peterson, M. R., and Scheller, R. H. (1993) Cell 72, 153-159 [Medline] [Order article via Infotrieve]
  61. Bommert, K., Charlton, M. P., DeBello, W. M., Chin, G. J., Betz, H., and Augustine, G. J. (1993) Nature 363, 163-165 [CrossRef][Medline] [Order article via Infotrieve]
  62. Nonet, M. L., Grundahl, K., Meyer, B. J., and Rand, J. B. (1993) Cell 73, 1291-1305 [Medline] [Order article via Infotrieve]
  63. DiAntonio, A., Parfit, K., and Schwarz, T. L. (1993) Cell 73, 1281-1290 [Medline] [Order article via Infotrieve]
  64. Geppert, M., Goda, Y., Hammer, R. E., Li, C., Rosahl, T. W., Stevens, C. F., and Südhof, T. C. (1994) Cell 79, 717-727 [Medline] [Order article via Infotrieve]
  65. Littleton, J. T., Stern, M., Schulze, K., Perin, M., and Bellen, H. J. (1993) Cell 74, 1125-1143 [Medline] [Order article via Infotrieve]
  66. DiAntonio, A., and Schwarz, T. L. (1994) Neuron 909-920
  67. Broadie, K., Bellen, H. J., DiAntonio, A., Littleton, J. T., and Schwarz, T. L. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10727-10731 [Abstract/Free Full Text]
  68. Reist, N. E., Buchanan, J., DiAntonio, A., and Schwarz, T. L. (1994) Soc. Neurosci. Abstr. 20, 199.10
  69. Zhang, J. Z., Davletov, B. A., Südhof, T. C., and Anderson, R. G. W. (1994) Cell 78, 751-760 [Medline] [Order article via Infotrieve]
  70. Martin, T. F. J., and Walent, J. H. (1989) J. Biol. Chem. 264, 10299-10308 [Abstract/Free Full Text]
  71. Hay, J. C., and Martin, T. F. (1992) J. Cell Biol. 119, 139-151 [Abstract]
  72. Walent, J. H., Porter, B. W., and Martin, T. F. J. (1992) Cell 70, 765-775 [Medline] [Order article via Infotrieve]
  73. Martin, T. F. J. (1994) Curr. Opin. Neurobiol. 4, in press
  74. Llinas, R., Steinberg, I. Z., and Walton, K. (1981) Biophys. J. 33, 323-351 [Abstract]
  75. Thomas, P., Wong, J. G., and Almers, W. (1993) EMBO J. 12, 303-306 [Abstract]
  76. Yoshida, A., Oho, C., Omori, A., Kuwahara, R., Ito, T., and Takahashi, M. (1992) J. Biol. Chem. 267, 24925-24928 [Abstract/Free Full Text]
  77. Gundersen, C. B., and Umbach, J. A. (1992) Neuron 9, 527-537 [Medline] [Order article via Infotrieve]
  78. Sapperstein, S. K., Walter, D. M., Grosvenor, A. R., Heuser, J. E., and Waters, M. G. (1995) Proc. Natl. Acad. Sci. U. S. A. , in press

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.