Biochemical Society Transactions
Phospholipid-binding proteins in calcium-dependent exocytosis Robert D. Burgoyne* and Alan Morgan The Physiological Laboratory, University of Liverpool, PO Box 147, Liverpool L69 3BX, U K
834
Introduction Exocytosis and other intracellular membrane fusions involve a specific interaction between transport vesicles and their target membranes, bilayer fusion and fission at a focal point. In the case of regulated exocytosis this process is triggered by intracellular second messengers such as CaL+.The various membrane fusion events that occur during vesicular traffic in cells must be controlled by proteins that confer specificity on the reaction and that modify lipid organization to lead to fusion and fission of lipid bilayers. Hilayer fusion in model systems can be driven by raising CaL+to millimolar levels but Ca’ -activated fusion at physiological (micromolar) Ca’ concentrations requires CaL+binding proteins that interact with phospholipid bilayers [ 11. One family of proteins that interact with phospholipids and are regulated by Ca2+ is the annexins [Z]. These proteins bind to phospholipids in a Ca’+-dependent manner and can stimulate membrane aggregation and fusion in in nitro systems [3-71. An alternative possible mechanism for Ca” activated membrane fusion is through stimulation of phospholipase A?. Hydrolysis of lipids by phospholipase A’ (PLA,) will generate free arachidonic acid and membrane lysophospholipids both of which are potentially fusogenic. In addition the ability of certain annexins to stimulate membrane fusion is greatly enhanced by unsaturated fatty acids such as arachidonate [S]. In this review we will assess the evidence that annexins and PLA, are involved in CaL+-activated membrane fusion, particularly in exocytosis from adrenal chromaffin cells. +
+
Annexins in exocytosis The annexin family consists of an increasing number of distinct but homologous CaL+- and phospholipid-binding proteins containing four to eight repeated conserved domains [2]. Certain annexins are apparently expressed ubiquitously in mammalian tissues while others have a more restricted distribution. Several of the annexins are able to bind to secretory vesicles in a Ca2+-dependent manner [8-111. Annexin I1 is also found as a tightly bound component of chromaffin granules
‘To whom correspondence should be addressed.
Volume 20
[ 121 and secretory granules from the anterior pituitary [13]. The ability of the annexins to aggregate phospholipid vesicles 13. 6. 7 ) and secretory vesicles [ 3 , 51 and to fuse secretory vesicles to each other [S] or to plasma membrane vesicles I 14, 15 I in vitro has led to the idea that they may be involved in Ca”-dependent membrane fusion in exocytosis in adrenal chromaffin cells [3* 5, 8, 0, 121, neutromammary phils [ 141, lung epithelial cells [ 1 epithelial cells [ 111, cardiac cells [ 101 and anterior pituitary cells [ 1.31. In addition, annexin I1 has been implicated in phagocytosis in neutrophils [ 161. In adrenal chromaffin cells, annexin I1 appears from several studies to be the annexin most likely to be involved in exocytosis. Annexin I1 is mainly localized on the inner surface of the plasma membrane in these cells [17, 181 and at lower levels on the chromaffin granule membrane [ 121. Several of the annexins are able to aggregate isolated chromaffin granules but only annexin I1 has been shown to do so at CaL+levels as low as those that activate exocytosis in permeabilized cells I 14 1. In this system Drust and Creutz 112) also showed that aggregated chromaffin granules will fuse after addition of arachidonic acid. The ability of annexin I1 to stimulate membrane fusion and its localization at relevant sites in the cell (plasma membrane and granules) would be consistent with this protein being involved in Ca’+-dependent exocytosis. This idea has been supported by functional studies on permeabilized chromaffin cells [ 19-21 1. Exocytotic release of catecholamines can be activated directly by introduction of micromolar Ca2+ into chromaffin cells permeabilized by digitonin [22]. Following permeabilization of the cells, their responsiveness to Ca‘+ runs down as they leak essential cytosolic components [ 19, 2.11. Annexin I1 is amongst the proteins that leak from the cells [21]. Introduction into the cells of purified annexin I1 (in its monomeric or heterotetrameric forms) results in increased CaL+-dependentsecretion but only at early times during run down [19-211. This suggests that leakage of annexin 11 contributes to but is not the sole cause of loss of responsiveness. Additional functional evidence that annexin I1 is involved in exocytosis came from the observation that a synthetic peptide based on the conserved annexin motif partially inhibited secretion in permeabilized chromaffin cells [ 101 and an
Lipid-Binding Proteins
antibody against the regulatory N-terminus of the protein enhanced Ca2+-dependent secretion [2 11. Our initial observations on annexin I1 effects in permeabilized chromaffin cells have been replicated by others [24,25] who have also shown that annexin I1 activity is enhanced by protein kinase C-mediated phosphorylation [24].
Phospholipase A, and arachidonic acid in exocytosis As noted above, chromafin granules aggregated by annexin I1 could be induced to fuse by addition of arachidonic acid. During chromaffin cell stimulation arachidonic acid is generated by a Ca‘+-dependent mechanism [25]. Since both arachidonic acid and lysophospholipids could be fusogenic it has been suggested that PLA, activation could be involved in the pathway leading to exocytosis [26-281. Treatment of plasma membranes with exogenous PLA, converted them into a fusogenic state such that chromaffin granules added to the plasma membrane fused with them [29] and released catecholFig. I Effect of bee venom phospholipase A, on Ca2+dependent secretion from permeabilized bovine adrenal chromaffin cells Chromaffin cells were pre-permeabilized with 20 , L ~ M digitonin [I91 for 6 min, and then challenged with 0 or 10 ,LtM-Ca2+with or without exogenous PLA, Catecholamine, released over I 5 min, was determined and expressed as a percentage of total cellular catecholamine The data shown are the calculated Ca2+-dependent secretion Catecholamine release at 0 Ca2+ was increased at the highest dose of PLA, used The PLA, was derived from bee venom (Sigma) In this experiment 5 mg/ml of fatty acid-free BSA was present throughout
24
1
U
2
20-
Q
Q)
L
16-
Q)
.-c
-E0 Q
c o
128 -
0) *,
Q 0
4 -
bQ
00
6
5
4
3
2
-log mg/ml phospholipase Az
amine [28]. In both studies arachidonic acid could be ruled out as being responsible for the generation of the fusogenic state. Investigation of the role of arachidonic acid in Ca2+-dependent exocytosis in permeabilized chromaffin cells also suggested that arachidonic acid did not drive membrane fusion [30, 311 since addition of exogenous arachidonic acid produced no more than a small increase in Ca2+-independent secretion [ 301. Various PLA, inhibitors failed to reduce Ca2+-dependent secretion and arachidonic acid release could be markedly inhibited without any major effect on secretion [31]. In addition, inclusion in permeabilization and stimulation buffers of 5 mg/ml of fatty acid-free BSA, to trap free arachidonic acid and extract membrane-associated free fatty acids, had no effect on the extent of CaZ+ -dependent secretion from chromafin cells [31]. These results suggest that generation of free arachidonic acid is not an essential step in the exocytotic pathway. The data does not, however, rule out the possibility that limited PLA, activity leading to lysophospholipid generation in plasma or secretory granule membranes may be involved in exocytosis. In fact, bee venom PLA, which was without effect on intact chromaffin cells, stimulated Ca2 -dependent secretion from permeabilized cells (Fig. 1). This finding does not necessarily mean that the exogenous PLA, is mimicking a physiological mechanism. +
Ex01 ( 14-3-3 protein) in exocytosis As noted above, addition of annexin I1 to permeabilized chromafin cells only stimulated secretion at early times of run down. This suggested that additional proteins required for exocytosis were lost from the cells. In an attempt to identify other cytosolic proteins involved in exocytosis we screened adrenal medullary and brain cytosols for factors active in the permeabilized cell/run down assay. Three factors were found, two stimulatory (Exol and Exo2) and one inhibitory [32]. Exol was purified to homogeneity and found to consist of multiple, approximately 30 kDa polypeptides. Sequence analysis demonstrated [32] that these were related to a family of previously known proteins called the 14-3-3 protein family [33-341. Exol is able to stimulate secretion even after prolonged run-down. The characteristics of its effects are fully consistent with stimulation of exocytosis since the stimulation is Ca2+- and MgATP-dependent [32, 351 and is blocked by tetanus toxin [36]. The mechanism by which Exol leads to a stimulation of Ca2+-dependent exocytosis is not
I992
835
Biochemical Society Transactions
836
known but an important clue has come from recent work on cytosolic I’l.A1. Platelet cytosolic PLA2 was purified and found to be highly activated by micromolar C a l f 1371. Recent cloning has shown that the cytosolic H,A? proteins are 14-3-3 proteins [ 381. T h e possibility therefore emerges that the effect of Ex01 on exocytosis is due to an intrinsic C a l f -dependent €‘LAL activity. T h e PLA2 activity of the 14-3-3 proteins has yet to be confirmed and its significance for exocytosis examined but this would certainly provide an explanation for the mode of action of E x o l . O n e speculative model for Calf -dependent exocytosis is that annexin I1 is responsible for the initial attachment of secretory granules to the exocytotic sites on the plasma membrane [ 181 where the €‘LA1 activity of Ex01 would generate fusogenic lysophospholipids. T h e relationship between Ex01 and annexin I1 (and Exo2) has yet to be established but current data suggest that these proteins play essential roles in exocytosis in chromaffin cells. This work was supported by a grant from The Wellcome Trust. 1. Meers, I)., Hong, K. 81 I’apaphodjopoulos, L). (1991) Ann. N.Y. Acad. Sci. 635,259-272 2. Hurgoyne, K. L). & (kisow, M. J. ( 1 989) Cell Calcium 10.1-10 3. Creutz. C. E.. I’azoles. C. J. 81 I’ollard, H. €3. (1078) J. Hiol. Chem. 253, 2858-2800 4. Sudhof. T. C.. Ebbecke, M.. Walker, J. H., Fritsche, LJ. & Houstead. C. (19x4) Biochemistry 23. 1103-1 109 5. Llrust, L). S. & Creutz. C. E. (1988) Nature 331, 88-91 6. I’owell, M. A. & Glenney, J. K. (19x7) Hiochem. J. 247, 321-328 7. Hlackwood, K. A. & Ernst, J. 1). (1990) Hiochem. J. 266, 195-200 8. Geisow. M. J. & Hurgoyne, K. L). (1982) J. Neurochem. 38, 1735-1741 9. Creutz, C. E., Dowling, I,. G., Sando, J. J., VillarI’alasi, C., Whipple, J. H. & Zaks, W. J. (1983) J. Hiol. Chem. 258, 14664-3 4674 10. Doubell, A. F., Rester. A. J. & Thibault. G. (1991) Hypertension 18, 648-650 11. Handel, S. E., Kennison, M. E., Wilde, C. J. & Hurgoyne, K. L). (1991) Cell Tissue Kes. 264, 549-554 12. Llrust, L). E. & Creutz, C. E. (1091) J. Neurochem. 56, 100-478 13. Turgeon, J. I,., Cooper, K. H. & Waring, L). W. (1990) Endocrinology 128, Oh- 102
Volume 20
14. Oshry, I,.* Meers, l’., Mealy, 7’. & Taubcr. A. I. (1001) Biochim. Biophys. Acta 1066,239-241 15. Chander, A. & Wu, K.-L). (1901) Hiochim. Hiophys. Acta 1086, 157-166 16. Ernst, J. I). (1991) J. Immunol. 146, 31 10-31 1 1 17. Burgoyne, K.D. & Cheek. T. K. (1087) Iliosci. Rep. 7. 281-288 18. Nakata, T., Sobue. K. & Hirokawa, N. (lOO0) J. Cell Biol. 110, 13-25 19. Ali, S. M., Geisow, M. J. 81 Hurgoyne, K. I). (10x0) Nature 340 3 13-3 15 20. Ali, S. M. & Hurgoyne. K. 1). (1000) Cell Signal 2. 265-276 21. Burgoyne, K. L). & Morgan. A. (1000) Hiochem. Soc. Trans. 18,1101-1 104 22. Dunn, 1,. A. & €Iolz, K. W. (1083) J. Ijiol. (‘hem. 258, 4989-4993 23. Sarafian, T., Aunis, 1). & Ilader, M. F. (1087) J. fliol. Chem. 262, 1667 1- 1Oh70 24. Sarafian, T., I’radel, I,.-A,, I lenry, J.-I’., Aunis, 1). & Hader, M-F. (1001) J. Cell Hiol. 114, 1135-1 147 25. Wu, Y. N. & Wagner, 1’. I). ( I O O I ) FEllS I,ett. 282, 197- 199 26. Frye, K. A. & Holz, K.W. (1085) J. Ncurochem. 44, 265-273 27. Moskowitz, N., Schook, W. & I’uszkin, S. (10x2) Science 216,305-307 28. Izumi, F.,Yanagihara, N., Wada, A,, Toyohira, Y. & Kobayashi, H. (1080) FBHS 1,ett. 196. 340-352 29. Karli, O., Schafer, 7’. & Burger, M. M. (1000) I’roc. Natl. Acad. Sci. 1J.S.A.87, 5012-5015 30. Morgan, A. & llurgoyne, K. I). (1000) 13iochem. J.
269,521-520 31. Morgan, A. & Hurgoyne, K. I). (1990) Hiochem. J. 271, 571-571 32. Morgan, A. & Hurgoyne. K. I). (1002) Nature 355, 83 3-836 33. Toker, A,, Ellis, C. A,, Sellers, I,. A. 81 Aitken, A. (1990) Eur. J. lliochem. 191, 421-420 34. Ichimura, T., Isobe, T., Okuyama, T., Takahashi, N., Araki, K., Kuwano, K. & Takahashi. Y. (1088) I’roc. Natl. Acad. Sci. LJ.S.A.85. 7084-7088 35. Morgan, A,, Hurgoyne, K.I). (lOO2) Hiochcm. J. 286, 807-8 1 1 36. Morgan, A.. Cenci de Hello. I.. Wcllcr, IT., 1)olly. J. 0. & Burgoyne, K. I). (1092) In Ilotulinum, tetanus neurotoxins: neurotransmission and biomedical aspects (Llasgupta, H., ed.), I’lenum I’ress, New York 37. Loeb, I,. A. & Gross, K. W. (1 080) J. lliol. Chem. 261, 10467-10470 38. Zupan, I,. A,, Steffens, I). I,., Berry, C. A,, l a d , M. & Gross, K.W. (1002)J. Iliol. Chem. 267, X707-X710 Received 14 July 1002
Phospholipid-binding proteins in calcium-dependent exocytosis Robert D. Burgoyne* and Alan Morgan The Physiological Laboratory, University of Liverpool, PO Box 147, Liverpool L69 3BX, U K
834
Introduction Exocytosis and other intracellular membrane fusions involve a specific interaction between transport vesicles and their target membranes, bilayer fusion and fission at a focal point. In the case of regulated exocytosis this process is triggered by intracellular second messengers such as CaL+.The various membrane fusion events that occur during vesicular traffic in cells must be controlled by proteins that confer specificity on the reaction and that modify lipid organization to lead to fusion and fission of lipid bilayers. Hilayer fusion in model systems can be driven by raising CaL+to millimolar levels but Ca’ -activated fusion at physiological (micromolar) Ca’ concentrations requires CaL+binding proteins that interact with phospholipid bilayers [ 11. One family of proteins that interact with phospholipids and are regulated by Ca2+ is the annexins [Z]. These proteins bind to phospholipids in a Ca’+-dependent manner and can stimulate membrane aggregation and fusion in in nitro systems [3-71. An alternative possible mechanism for Ca” activated membrane fusion is through stimulation of phospholipase A?. Hydrolysis of lipids by phospholipase A’ (PLA,) will generate free arachidonic acid and membrane lysophospholipids both of which are potentially fusogenic. In addition the ability of certain annexins to stimulate membrane fusion is greatly enhanced by unsaturated fatty acids such as arachidonate [S]. In this review we will assess the evidence that annexins and PLA, are involved in CaL+-activated membrane fusion, particularly in exocytosis from adrenal chromaffin cells. +
+
Annexins in exocytosis The annexin family consists of an increasing number of distinct but homologous CaL+- and phospholipid-binding proteins containing four to eight repeated conserved domains [2]. Certain annexins are apparently expressed ubiquitously in mammalian tissues while others have a more restricted distribution. Several of the annexins are able to bind to secretory vesicles in a Ca2+-dependent manner [8-111. Annexin I1 is also found as a tightly bound component of chromaffin granules
‘To whom correspondence should be addressed.
Volume 20
[ 121 and secretory granules from the anterior pituitary [13]. The ability of the annexins to aggregate phospholipid vesicles 13. 6. 7 ) and secretory vesicles [ 3 , 51 and to fuse secretory vesicles to each other [S] or to plasma membrane vesicles I 14, 15 I in vitro has led to the idea that they may be involved in Ca”-dependent membrane fusion in exocytosis in adrenal chromaffin cells [3* 5, 8, 0, 121, neutromammary phils [ 141, lung epithelial cells [ 1 epithelial cells [ 111, cardiac cells [ 101 and anterior pituitary cells [ 1.31. In addition, annexin I1 has been implicated in phagocytosis in neutrophils [ 161. In adrenal chromaffin cells, annexin I1 appears from several studies to be the annexin most likely to be involved in exocytosis. Annexin I1 is mainly localized on the inner surface of the plasma membrane in these cells [17, 181 and at lower levels on the chromaffin granule membrane [ 121. Several of the annexins are able to aggregate isolated chromaffin granules but only annexin I1 has been shown to do so at CaL+levels as low as those that activate exocytosis in permeabilized cells I 14 1. In this system Drust and Creutz 112) also showed that aggregated chromaffin granules will fuse after addition of arachidonic acid. The ability of annexin I1 to stimulate membrane fusion and its localization at relevant sites in the cell (plasma membrane and granules) would be consistent with this protein being involved in Ca’+-dependent exocytosis. This idea has been supported by functional studies on permeabilized chromaffin cells [ 19-21 1. Exocytotic release of catecholamines can be activated directly by introduction of micromolar Ca2+ into chromaffin cells permeabilized by digitonin [22]. Following permeabilization of the cells, their responsiveness to Ca‘+ runs down as they leak essential cytosolic components [ 19, 2.11. Annexin I1 is amongst the proteins that leak from the cells [21]. Introduction into the cells of purified annexin I1 (in its monomeric or heterotetrameric forms) results in increased CaL+-dependentsecretion but only at early times during run down [19-211. This suggests that leakage of annexin 11 contributes to but is not the sole cause of loss of responsiveness. Additional functional evidence that annexin I1 is involved in exocytosis came from the observation that a synthetic peptide based on the conserved annexin motif partially inhibited secretion in permeabilized chromaffin cells [ 101 and an
Lipid-Binding Proteins
antibody against the regulatory N-terminus of the protein enhanced Ca2+-dependent secretion [2 11. Our initial observations on annexin I1 effects in permeabilized chromaffin cells have been replicated by others [24,25] who have also shown that annexin I1 activity is enhanced by protein kinase C-mediated phosphorylation [24].
Phospholipase A, and arachidonic acid in exocytosis As noted above, chromafin granules aggregated by annexin I1 could be induced to fuse by addition of arachidonic acid. During chromaffin cell stimulation arachidonic acid is generated by a Ca‘+-dependent mechanism [25]. Since both arachidonic acid and lysophospholipids could be fusogenic it has been suggested that PLA, activation could be involved in the pathway leading to exocytosis [26-281. Treatment of plasma membranes with exogenous PLA, converted them into a fusogenic state such that chromaffin granules added to the plasma membrane fused with them [29] and released catecholFig. I Effect of bee venom phospholipase A, on Ca2+dependent secretion from permeabilized bovine adrenal chromaffin cells Chromaffin cells were pre-permeabilized with 20 , L ~ M digitonin [I91 for 6 min, and then challenged with 0 or 10 ,LtM-Ca2+with or without exogenous PLA, Catecholamine, released over I 5 min, was determined and expressed as a percentage of total cellular catecholamine The data shown are the calculated Ca2+-dependent secretion Catecholamine release at 0 Ca2+ was increased at the highest dose of PLA, used The PLA, was derived from bee venom (Sigma) In this experiment 5 mg/ml of fatty acid-free BSA was present throughout
24
1
U
2
20-
Q
Q)
L
16-
Q)
.-c
-E0 Q
c o
128 -
0) *,
Q 0
4 -
bQ
00
6
5
4
3
2
-log mg/ml phospholipase Az
amine [28]. In both studies arachidonic acid could be ruled out as being responsible for the generation of the fusogenic state. Investigation of the role of arachidonic acid in Ca2+-dependent exocytosis in permeabilized chromaffin cells also suggested that arachidonic acid did not drive membrane fusion [30, 311 since addition of exogenous arachidonic acid produced no more than a small increase in Ca2+-independent secretion [ 301. Various PLA, inhibitors failed to reduce Ca2+-dependent secretion and arachidonic acid release could be markedly inhibited without any major effect on secretion [31]. In addition, inclusion in permeabilization and stimulation buffers of 5 mg/ml of fatty acid-free BSA, to trap free arachidonic acid and extract membrane-associated free fatty acids, had no effect on the extent of CaZ+ -dependent secretion from chromafin cells [31]. These results suggest that generation of free arachidonic acid is not an essential step in the exocytotic pathway. The data does not, however, rule out the possibility that limited PLA, activity leading to lysophospholipid generation in plasma or secretory granule membranes may be involved in exocytosis. In fact, bee venom PLA, which was without effect on intact chromaffin cells, stimulated Ca2 -dependent secretion from permeabilized cells (Fig. 1). This finding does not necessarily mean that the exogenous PLA, is mimicking a physiological mechanism. +
Ex01 ( 14-3-3 protein) in exocytosis As noted above, addition of annexin I1 to permeabilized chromafin cells only stimulated secretion at early times of run down. This suggested that additional proteins required for exocytosis were lost from the cells. In an attempt to identify other cytosolic proteins involved in exocytosis we screened adrenal medullary and brain cytosols for factors active in the permeabilized cell/run down assay. Three factors were found, two stimulatory (Exol and Exo2) and one inhibitory [32]. Exol was purified to homogeneity and found to consist of multiple, approximately 30 kDa polypeptides. Sequence analysis demonstrated [32] that these were related to a family of previously known proteins called the 14-3-3 protein family [33-341. Exol is able to stimulate secretion even after prolonged run-down. The characteristics of its effects are fully consistent with stimulation of exocytosis since the stimulation is Ca2+- and MgATP-dependent [32, 351 and is blocked by tetanus toxin [36]. The mechanism by which Exol leads to a stimulation of Ca2+-dependent exocytosis is not
I992
835
Biochemical Society Transactions
836
known but an important clue has come from recent work on cytosolic I’l.A1. Platelet cytosolic PLA2 was purified and found to be highly activated by micromolar C a l f 1371. Recent cloning has shown that the cytosolic H,A? proteins are 14-3-3 proteins [ 381. T h e possibility therefore emerges that the effect of Ex01 on exocytosis is due to an intrinsic C a l f -dependent €‘LAL activity. T h e PLA2 activity of the 14-3-3 proteins has yet to be confirmed and its significance for exocytosis examined but this would certainly provide an explanation for the mode of action of E x o l . O n e speculative model for Calf -dependent exocytosis is that annexin I1 is responsible for the initial attachment of secretory granules to the exocytotic sites on the plasma membrane [ 181 where the €‘LA1 activity of Ex01 would generate fusogenic lysophospholipids. T h e relationship between Ex01 and annexin I1 (and Exo2) has yet to be established but current data suggest that these proteins play essential roles in exocytosis in chromaffin cells. This work was supported by a grant from The Wellcome Trust. 1. Meers, I)., Hong, K. 81 I’apaphodjopoulos, L). (1991) Ann. N.Y. Acad. Sci. 635,259-272 2. Hurgoyne, K. L). & (kisow, M. J. ( 1 989) Cell Calcium 10.1-10 3. Creutz. C. E.. I’azoles. C. J. 81 I’ollard, H. €3. (1078) J. Hiol. Chem. 253, 2858-2800 4. Sudhof. T. C.. Ebbecke, M.. Walker, J. H., Fritsche, LJ. & Houstead. C. (19x4) Biochemistry 23. 1103-1 109 5. Llrust, L). S. & Creutz. C. E. (1988) Nature 331, 88-91 6. I’owell, M. A. & Glenney, J. K. (19x7) Hiochem. J. 247, 321-328 7. Hlackwood, K. A. & Ernst, J. 1). (1990) Hiochem. J. 266, 195-200 8. Geisow. M. J. & Hurgoyne, K. L). (1982) J. Neurochem. 38, 1735-1741 9. Creutz, C. E., Dowling, I,. G., Sando, J. J., VillarI’alasi, C., Whipple, J. H. & Zaks, W. J. (1983) J. Hiol. Chem. 258, 14664-3 4674 10. Doubell, A. F., Rester. A. J. & Thibault. G. (1991) Hypertension 18, 648-650 11. Handel, S. E., Kennison, M. E., Wilde, C. J. & Hurgoyne, K. L). (1991) Cell Tissue Kes. 264, 549-554 12. Llrust, L). E. & Creutz, C. E. (1091) J. Neurochem. 56, 100-478 13. Turgeon, J. I,., Cooper, K. H. & Waring, L). W. (1990) Endocrinology 128, Oh- 102
Volume 20
14. Oshry, I,.* Meers, l’., Mealy, 7’. & Taubcr. A. I. (1001) Biochim. Biophys. Acta 1066,239-241 15. Chander, A. & Wu, K.-L). (1901) Hiochim. Hiophys. Acta 1086, 157-166 16. Ernst, J. I). (1991) J. Immunol. 146, 31 10-31 1 1 17. Burgoyne, K.D. & Cheek. T. K. (1087) Iliosci. Rep. 7. 281-288 18. Nakata, T., Sobue. K. & Hirokawa, N. (lOO0) J. Cell Biol. 110, 13-25 19. Ali, S. M., Geisow, M. J. 81 Hurgoyne, K. I). (10x0) Nature 340 3 13-3 15 20. Ali, S. M. & Hurgoyne. K. 1). (1000) Cell Signal 2. 265-276 21. Burgoyne, K. L). & Morgan. A. (1000) Hiochem. Soc. Trans. 18,1101-1 104 22. Dunn, 1,. A. & €Iolz, K. W. (1083) J. Ijiol. (‘hem. 258, 4989-4993 23. Sarafian, T., Aunis, 1). & Ilader, M. F. (1087) J. fliol. Chem. 262, 1667 1- 1Oh70 24. Sarafian, T., I’radel, I,.-A,, I lenry, J.-I’., Aunis, 1). & Hader, M-F. (1001) J. Cell Hiol. 114, 1135-1 147 25. Wu, Y. N. & Wagner, 1’. I). ( I O O I ) FEllS I,ett. 282, 197- 199 26. Frye, K. A. & Holz, K.W. (1085) J. Ncurochem. 44, 265-273 27. Moskowitz, N., Schook, W. & I’uszkin, S. (10x2) Science 216,305-307 28. Izumi, F.,Yanagihara, N., Wada, A,, Toyohira, Y. & Kobayashi, H. (1080) FBHS 1,ett. 196. 340-352 29. Karli, O., Schafer, 7’. & Burger, M. M. (1000) I’roc. Natl. Acad. Sci. 1J.S.A.87, 5012-5015 30. Morgan, A. & llurgoyne, K. I). (1000) 13iochem. J.
269,521-520 31. Morgan, A. & Hurgoyne, K. I). (1990) Hiochem. J. 271, 571-571 32. Morgan, A. & Hurgoyne. K. I). (1002) Nature 355, 83 3-836 33. Toker, A,, Ellis, C. A,, Sellers, I,. A. 81 Aitken, A. (1990) Eur. J. lliochem. 191, 421-420 34. Ichimura, T., Isobe, T., Okuyama, T., Takahashi, N., Araki, K., Kuwano, K. & Takahashi. Y. (1088) I’roc. Natl. Acad. Sci. LJ.S.A.85. 7084-7088 35. Morgan, A,, Hurgoyne, K.I). (lOO2) Hiochcm. J. 286, 807-8 1 1 36. Morgan, A.. Cenci de Hello. I.. Wcllcr, IT., 1)olly. J. 0. & Burgoyne, K. I). (1092) In Ilotulinum, tetanus neurotoxins: neurotransmission and biomedical aspects (Llasgupta, H., ed.), I’lenum I’ress, New York 37. Loeb, I,. A. & Gross, K. W. (1 080) J. lliol. Chem. 261, 10467-10470 38. Zupan, I,. A,, Steffens, I). I,., Berry, C. A,, l a d , M. & Gross, K.W. (1002)J. Iliol. Chem. 267, X707-X710 Received 14 July 1002
