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Development of a cell-free model for compound exocytosis using components of the chromaffin cell
Journal of the Autonomic Nervous System, 7 (1983) 13-18
13
Elsevier Biomedical Press
Development of a cell-free model for compound exocytosis using components of the chromaffin cell C a r l E. C r e u t z a n d H a r v e y B. P o l l a r d Department of Pharmacology, University of Virginia, Charlottesville, VA 22908 and Laboratory of Cell Biology, National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases, Bethesda, MD 20205 (U.S.A.)
(Received June 1st, 1982) (Accepted July 20th, 1982)
K e y words: compound exocytosis--chromaffin c e l l - - C a 2÷ - - s y n e x i n - - m e m b r a n e
fusion
Abstract A study of exocytosis and the possible role of chromaffin granules in adrenal medulla secretion is presented. This review describes a system that may recreate Ca 2 ÷-dependent membrane contact and fusion events that occur in exocytosis. The nature and role of synexin and other compounds in this process are discussed.
Introduction We have been trying to understand the molecular events that occur during the release of catecholamines and proteins by exocytosis from the adrenal medullary chromaffin cell. Since exocytosis is a complex process involving many steps, our approach has been to isolate components of the secretory cells and recombine them in model systems that recreate one or two of the crucial steps in the overall process. This review will describe the development of a model system that appears to recreate Ca 2+ -dependent membrane contact and fusion events that occur in exocytosis.
Discovery and isolation of synexin, a possible receptor for
Ca 2 +
in exocytosis
Electron micrographs of stimulated chromaffin cells have revealed that during prolonged secretion compound exocytosis occurs, in which chromaffin granules fuse with one another in the cytoplasm, as well as to the plasma membrane, thus leading 0165-1838/83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press
14 to the formation of large vacuoles and channels through which secretory product is released [12]. This observation suggests that a study of the interaction of isolated chromaffin granules might provide a useful model for events occurring during compound exocytosis. However, isolated granules have been found to have a negative surface charge and tend not to interact unless bathed in high (unphysiologic) concentrations of divalent cations. We reasoned that during isolation of the granules some other component of the cell was lost that might mediate membrane interaction. Indeed, we found that the soluble fraction of the adrenal medullary homogenate would initiate contact between chromaffin granules [3]. This contact was manifested as an increase in the turbidity of a granule suspension as the granules formed large aggregates that could be seen in the phase-microscope. The aggregation phenomenon was dependent specifically on Ca 2+ and did not occur in the cold. Using this Ca 2+ and temperature-dependent increase in the turbidity of a granule suspension as an assay, we developed a scheme for the isolation of a single factor from the post-microsomal supernatant of the homogenate which was responsible for the aggregation [3]. This factor was a 47,000 dalton protein that we named synexin from the Greek 'synexis' (meaning a meeting) because of the meetings it initated between granule membranes.
Characteristics of synexin and its interaction with chromaffin granules A careful analysis of the C a 2+ dependence of synexin action on a granule suspension suggested that Ca 2+ was binding to a site with a dissociation constant of 200/~m [3]. Although this is an unusually high value for a cytoplasmic Ca z+ binding protein, we think its significance may be that it is intermediate between resting cytoplasmic Ca z+ concentrations of less than 10 - 6 M and extracellular concentrations of about 10 -3 M. Therefore, this process of membrane contact could only be activated near the surface membrane of the cell where the Ca 2+ enters through specific channels. This seems a reasonable design, if the purpose of synexin is to attach mature secretory vesicles with the plasma membrane, and not cause indiscriminate membrane contact throughout the cell. In subsequent studies on isolated synexin we found that C a 2+ caused a rapid polymerization of synexin into 5 by 15 nm rods [4]. These rods spontaneously assembled into regular bundles with their long axes aligned. In the presence of granule membranes, the rods or bundles bound to the membranes in a Ca 2+dependent fashion [3]. These events hint at a mechanism of membrane aggregation by synexin: perhaps the protein binds to sites on the granule membranes and then, by self-associating to form a rod, pulls the membranes together. At the moment, we can only speculate on such details of synexin action.
15
Generalization of synexin action In an important study which greatly influenced our thinking, Morris and Hughes discovered that synexin would aggregate liposomes composed of acidic phospholipids [10]. This observation was subsequently confirmed in studies by Hong et al. [9] who also demonstrated that the rate of fusion of acidic phospholiposomes in the presence of Ca2+ was accelerated by synexin. These observations were suprising since glutaraldehyde [3] or protease treatment [7] of granules had been found to inhibit the ability of synexin to cause aggregation, suggesting there might be a protein receptor for synexin on the granule surface. Furthermore, under similar incubation conditions synexin shows a specificity for granule membranes over mitochondrial membranes [3,6] and chromaffin cell plasma membranes over red blood cell membranes [14]. Therefore, the mode of attachment of synexin to a natural membrane and the cause of its varied specificity of action remain an important area of study. That synexin must perform a more general function than interaction with granule membranes is suggested by the apparent ubiquity of synexin. We have now obtained synexin-like preparations from the adrenal medulla and cortex [3], brain [3], parotid (C.E. Creutz, unpublished observation), liver [6] and spleen (J.H. Scott, unpublished observation) and from 3 species: cow [3], rat (C.E. Creutz and H.B. Pollard, unpublished observation) and rabbit (C.E. Creutz, unpublished observation). We also obtained a granule-aggregating activity from human platelets, but the physical properties of this factor were distinct from synexin and need to be explored further. All of these preparations were assayed with bovine chromaffin granules, but, in situ, they must interact with other specialized membranes, perhaps to promote exocytosis or membrane contact events of some other type. Between tissues and species we have found that the physical properties and Ca2+ dependence of the protein has been highly conserved (with the exception of the material from human platelets which is larger and more Ca 2÷ -sensitive; C.E. Creutz and H.B. Pollard, unpublished observations).
Membrane fusion following contact mediated by synexin Examination by means of the electron microscope of granule aggregates formed by synexin revealed that the membranes of the granules had been tightly sealed together to form "pentalaminar" junctions similar to those that have been observed between secretory vesicles and the plasma membranes of stimulated secretory cells [3]. However, there was no evidence of actual fusion of granule membranes to form larger structures, as would be anticipated if the model system correctly reflected events occurring in exocytosis. It appears that synexin alone is capable only of initiating contacts between membranes. Other factors present in the stimulated cell must be responsible for complete membrane fusion. One possible factor which does become available during exocytosis in the platelet and the chromaffin cell and probably other cells is free arachidonic acid, cleaved from phospholipids by Ca2+-
16 sensitive phospholipase A2. We were delighted to find that if we added arachidonic acid to granules that had been aggregated by synexin they very rapidly fused [2]. The concentrations necessary to cause this event were the same as those that occur in stimulated secretory cells (4% relative to total lipid). Other cis-unsaturated fatty acids that occur in natural membranes, such as oleic, linoleic and linolenic, also caused fusion, but their effectiveness decreased as their degree of saturation increased. Other types of lipids, such as trans-unsaturated fatty acids, saturated fatty acids, esterified cis-unsaturated fatty acids, lysolecithin, or analogues of oleic acid with the position of the double band displaced towards or away from the head group, were all ineffective in causing fusion [2]. When the granules did fuse they retained their soluble protein content, which appeared as a fibriller mesh in electron micrographs, but about 50% of their epinephrine was lost [2]. Perhaps the most striking feature of the fusion event was that the clumps of aggregated vesicles swelled to form large vacuole-like structures, some as large as 10 t~M in diameter, resulting from the fusion of 1600 chromaffin granules.
A chemiosmotic driving force for membrane fusion? We have wondered what causes the swelling of the fusing granules and, further, whether the tendency to swell might be the driving force behind the fusion event. One possibility we considered was the well-documented chemiosmotic swelling that granules undergo in the presence of MgATP and a permeant anion [1,5,11]. As a consequence of the inward pumping of protons by a membrane ATPase and the concurrent influx of a permeant anion such as CI- in response to the membrane potential generated by the ATPase, there is an osmotic pressure increase in the granule. In the case of isolated granules, since the membrane cannot appreciably stretch, the granules burst, releasing their soluble contents. If two granules were attached to one another by synexin, and the point of contact were specifically weakened by arachidonic acid, a pressure increase within the granules might be relaxed by breaking the pentalaminar contact point and fusing the two granules to form a larger vesicle with a smaller surface-to-volume ratio. In the case of our model system using granules, synexin and arachidonic acid, some type of expansion of the core matrix must have been sufficient to drive fusion, since the event was not dependent on MgATP or C1-. However, in the structured environment of the cytoplasm, expansion of the granules might be more difficult and require chemiosmotic forces. We cling to the view that such chemiosmotic swelling may be important in the living cell beause it is a view that has correctly predicted the behavior of seretory cells under certain circumstances. For example, secretion from cells can be blocked by increased osmotic strength of the culture medium, by pharmacological blockade of anion transport, or by proton ionophores (which short-circuit the proton pump), just as would be predicted if chemiosmotic swelling of granules were important in secretion [12,13].
17
Summary: conceptual integration of the model systems Fig. 1 presents a summary picture of the way in which the model system we have developed for membrane contact and fusion may fit into the overall mechanism of exocytosis in a cell. The figure also incorporates another model system for events before membrane fusion involving release of the granule from an actin-containing cytoskeleton [8], as explained in the legend. The overall scheme is a summation of individual steps that we have been able to recreate in the test tube. Are these steps in vitro artifacts, or do they really occur in the cell? This question poses the greatest challenge to us as model builders.
MODEL FOR PROCESS OF EXOCYTOSlS FROM CHROMAFFIN CELLS
co poun, Exocytosis[
Cytoskeleton
ctin,
\
~
Synexin,
(( isethionat~~FP,
)/ Pentalaminar Complex
/
///
,FP PMTHZ
Fig. l. Model for the process of exocytosis from chromaffin ceils. The events occurring during exocytosis are numbered in a temporal sequence, perhaps related to the increase in the free calcium concentration within the cell after some stimulus. Reaction 1 involves the Ca2+-dependent dissociation of the granule membrane from its cross-linked state with cytoskeletal actin, as the free calcium concentration rises above 10 -6 M. Thus, the secretory granule becomes specifically dislocated from its position in the cytoplasm. Subsequently, as the calcium concentration rises towards 10-5 M near the plasma membrane, reaction 2 becomes dominant and causes the secretory granule to become specifically attached to the inner aspect of the plasma membrane by means of Ca2~--activated synexin. This process is approximately equally sensitive to both the phenothiazine drugs trifluoperazine (TFP) and promethazine (PMTHZ) [6]. Following attachment, we presume that a granule-based osmotic gradient develops, possibly chemiosmotic in origin, and arachidonic acid release occurs. Both processes are represented by reaction 3, because at present we do not know the timing or the relative importance of the two. We presume that compound exocytosis can then occur, in which a free, intact granule can undergo reactions 2 and 3 with the granule membrane remnant of a past exocytotic event.
18
Acknowledgements Unpublished work described here on rabbit synexin was supported by Biomedical Research Support Grant 5 S07 RR05431-20 to the University of Virginia School of Medicine. We thank Shirley Davis for typing this manuscript.
References 1 Casey, R.P., Njus, O., Radda, G.K., and Sehr, P.A., ATP-evoked catecholamine release in chromaffin granules: osmotic lysis as a consequence of proton translation, Biochem. J., 158 (1976) 583-588. 2 Creutz, C.E., Cis-unsaturated fatty acids induce the fusion of chromaffin granules aggregated by synexin, J. Cell. Biol. 91 (1981) 247-256. 3 Creutz, C.E., Pazoles, C.J. and Pollard, H.B., Identification and purification of an adrenal medullary protein (synexin) that causes calcium dependent aggregation of isolated chromaffin granules, J. biol. Chem., 253 (1978) 2858-2866. 4 Creutz, C.E., Pazoles, C.J. and Pollard, H.B., Self-association of synexin in the presence of calcium, J. biol. Chem., 254 (1979) 553-558. 5 Creutz, C.E. and Pollard, H.B., A biophysical model of the chromaffin granule: accurate description of the kinetics of ATP and Cl--dependent granule lysis, Biophys. J. 31 (1980) 255 270. 6 Creutz, C.E., Scott, J.H., Pazoles, C.J. and Pollard, H.B., Further characterization of the aggregation and fusion of chromaffin granules by synexin as a model for compound exocytosis, J. cell. Biochem.. 18 (1982) 87-97. 7 Dabrow, M., Zaremba, S. and Hogue-Angeletti, R.A., Specificity of synexin-induced chromaffin granule aggregation, Biochem. Biophys. Res. Commun., 96 (1980) 1164-1171. 8 Fowler, V.M. and Pollard, H.B., Chromaffin granule membrane-F actin interactions are Ca 2+ sensitive, Nature (Lond.), 295 (1982) 336-339. 9 Hong, K., Duzgunes, N. and Papahadjopoulos, D., Role of synexin in membrane fusion, J. biol. Chem., 256 (1981) 3641-3644. 10 Morris, S.J. and Hughes J.M.X., Synexin protein is nonselective in its ability to increase Ca 2+dependent aggregation of biological and artifical membranes, Biochem. Biophys. Res. Commun., 91 (1979) 345-350. 11 Pazoles, C.J. and Pollard, H.B., Evidence for stimulation of anion transport in ATP-evoked transmitter release from isolated secretory vesicles, J. biol. Chem. 253 (1978) 3962-3969. 12 Pollard, H.B., Pazoles, C.J. and Creutz, C.E., Mechanism of calcium action and release of vesicle-bound hormones during exocytosis, Recent Progr. Horm. Res., 37 (1981) 299-332. 13 Pollard, H.B., Pazoles, C.J., Creutz, C.E. and Zinder, O., The chromaffin granule and possible mechanisms of exocytosis, Int. Rev. Cytol., 58 (1979) 160-198. 14 Scott, J.H., Creutz, C.E. and Pollard, H.B., Synexin binding to chromaffin cell plasma membrane. Europ. J. Cell. Biol., 22 (1980) 186 (Abstr.).
13
Elsevier Biomedical Press
Development of a cell-free model for compound exocytosis using components of the chromaffin cell C a r l E. C r e u t z a n d H a r v e y B. P o l l a r d Department of Pharmacology, University of Virginia, Charlottesville, VA 22908 and Laboratory of Cell Biology, National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases, Bethesda, MD 20205 (U.S.A.)
(Received June 1st, 1982) (Accepted July 20th, 1982)
K e y words: compound exocytosis--chromaffin c e l l - - C a 2÷ - - s y n e x i n - - m e m b r a n e
fusion
Abstract A study of exocytosis and the possible role of chromaffin granules in adrenal medulla secretion is presented. This review describes a system that may recreate Ca 2 ÷-dependent membrane contact and fusion events that occur in exocytosis. The nature and role of synexin and other compounds in this process are discussed.
Introduction We have been trying to understand the molecular events that occur during the release of catecholamines and proteins by exocytosis from the adrenal medullary chromaffin cell. Since exocytosis is a complex process involving many steps, our approach has been to isolate components of the secretory cells and recombine them in model systems that recreate one or two of the crucial steps in the overall process. This review will describe the development of a model system that appears to recreate Ca 2+ -dependent membrane contact and fusion events that occur in exocytosis.
Discovery and isolation of synexin, a possible receptor for
Ca 2 +
in exocytosis
Electron micrographs of stimulated chromaffin cells have revealed that during prolonged secretion compound exocytosis occurs, in which chromaffin granules fuse with one another in the cytoplasm, as well as to the plasma membrane, thus leading 0165-1838/83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press
14 to the formation of large vacuoles and channels through which secretory product is released [12]. This observation suggests that a study of the interaction of isolated chromaffin granules might provide a useful model for events occurring during compound exocytosis. However, isolated granules have been found to have a negative surface charge and tend not to interact unless bathed in high (unphysiologic) concentrations of divalent cations. We reasoned that during isolation of the granules some other component of the cell was lost that might mediate membrane interaction. Indeed, we found that the soluble fraction of the adrenal medullary homogenate would initiate contact between chromaffin granules [3]. This contact was manifested as an increase in the turbidity of a granule suspension as the granules formed large aggregates that could be seen in the phase-microscope. The aggregation phenomenon was dependent specifically on Ca 2+ and did not occur in the cold. Using this Ca 2+ and temperature-dependent increase in the turbidity of a granule suspension as an assay, we developed a scheme for the isolation of a single factor from the post-microsomal supernatant of the homogenate which was responsible for the aggregation [3]. This factor was a 47,000 dalton protein that we named synexin from the Greek 'synexis' (meaning a meeting) because of the meetings it initated between granule membranes.
Characteristics of synexin and its interaction with chromaffin granules A careful analysis of the C a 2+ dependence of synexin action on a granule suspension suggested that Ca 2+ was binding to a site with a dissociation constant of 200/~m [3]. Although this is an unusually high value for a cytoplasmic Ca z+ binding protein, we think its significance may be that it is intermediate between resting cytoplasmic Ca z+ concentrations of less than 10 - 6 M and extracellular concentrations of about 10 -3 M. Therefore, this process of membrane contact could only be activated near the surface membrane of the cell where the Ca 2+ enters through specific channels. This seems a reasonable design, if the purpose of synexin is to attach mature secretory vesicles with the plasma membrane, and not cause indiscriminate membrane contact throughout the cell. In subsequent studies on isolated synexin we found that C a 2+ caused a rapid polymerization of synexin into 5 by 15 nm rods [4]. These rods spontaneously assembled into regular bundles with their long axes aligned. In the presence of granule membranes, the rods or bundles bound to the membranes in a Ca 2+dependent fashion [3]. These events hint at a mechanism of membrane aggregation by synexin: perhaps the protein binds to sites on the granule membranes and then, by self-associating to form a rod, pulls the membranes together. At the moment, we can only speculate on such details of synexin action.
15
Generalization of synexin action In an important study which greatly influenced our thinking, Morris and Hughes discovered that synexin would aggregate liposomes composed of acidic phospholipids [10]. This observation was subsequently confirmed in studies by Hong et al. [9] who also demonstrated that the rate of fusion of acidic phospholiposomes in the presence of Ca2+ was accelerated by synexin. These observations were suprising since glutaraldehyde [3] or protease treatment [7] of granules had been found to inhibit the ability of synexin to cause aggregation, suggesting there might be a protein receptor for synexin on the granule surface. Furthermore, under similar incubation conditions synexin shows a specificity for granule membranes over mitochondrial membranes [3,6] and chromaffin cell plasma membranes over red blood cell membranes [14]. Therefore, the mode of attachment of synexin to a natural membrane and the cause of its varied specificity of action remain an important area of study. That synexin must perform a more general function than interaction with granule membranes is suggested by the apparent ubiquity of synexin. We have now obtained synexin-like preparations from the adrenal medulla and cortex [3], brain [3], parotid (C.E. Creutz, unpublished observation), liver [6] and spleen (J.H. Scott, unpublished observation) and from 3 species: cow [3], rat (C.E. Creutz and H.B. Pollard, unpublished observation) and rabbit (C.E. Creutz, unpublished observation). We also obtained a granule-aggregating activity from human platelets, but the physical properties of this factor were distinct from synexin and need to be explored further. All of these preparations were assayed with bovine chromaffin granules, but, in situ, they must interact with other specialized membranes, perhaps to promote exocytosis or membrane contact events of some other type. Between tissues and species we have found that the physical properties and Ca2+ dependence of the protein has been highly conserved (with the exception of the material from human platelets which is larger and more Ca 2÷ -sensitive; C.E. Creutz and H.B. Pollard, unpublished observations).
Membrane fusion following contact mediated by synexin Examination by means of the electron microscope of granule aggregates formed by synexin revealed that the membranes of the granules had been tightly sealed together to form "pentalaminar" junctions similar to those that have been observed between secretory vesicles and the plasma membranes of stimulated secretory cells [3]. However, there was no evidence of actual fusion of granule membranes to form larger structures, as would be anticipated if the model system correctly reflected events occurring in exocytosis. It appears that synexin alone is capable only of initiating contacts between membranes. Other factors present in the stimulated cell must be responsible for complete membrane fusion. One possible factor which does become available during exocytosis in the platelet and the chromaffin cell and probably other cells is free arachidonic acid, cleaved from phospholipids by Ca2+-
16 sensitive phospholipase A2. We were delighted to find that if we added arachidonic acid to granules that had been aggregated by synexin they very rapidly fused [2]. The concentrations necessary to cause this event were the same as those that occur in stimulated secretory cells (4% relative to total lipid). Other cis-unsaturated fatty acids that occur in natural membranes, such as oleic, linoleic and linolenic, also caused fusion, but their effectiveness decreased as their degree of saturation increased. Other types of lipids, such as trans-unsaturated fatty acids, saturated fatty acids, esterified cis-unsaturated fatty acids, lysolecithin, or analogues of oleic acid with the position of the double band displaced towards or away from the head group, were all ineffective in causing fusion [2]. When the granules did fuse they retained their soluble protein content, which appeared as a fibriller mesh in electron micrographs, but about 50% of their epinephrine was lost [2]. Perhaps the most striking feature of the fusion event was that the clumps of aggregated vesicles swelled to form large vacuole-like structures, some as large as 10 t~M in diameter, resulting from the fusion of 1600 chromaffin granules.
A chemiosmotic driving force for membrane fusion? We have wondered what causes the swelling of the fusing granules and, further, whether the tendency to swell might be the driving force behind the fusion event. One possibility we considered was the well-documented chemiosmotic swelling that granules undergo in the presence of MgATP and a permeant anion [1,5,11]. As a consequence of the inward pumping of protons by a membrane ATPase and the concurrent influx of a permeant anion such as CI- in response to the membrane potential generated by the ATPase, there is an osmotic pressure increase in the granule. In the case of isolated granules, since the membrane cannot appreciably stretch, the granules burst, releasing their soluble contents. If two granules were attached to one another by synexin, and the point of contact were specifically weakened by arachidonic acid, a pressure increase within the granules might be relaxed by breaking the pentalaminar contact point and fusing the two granules to form a larger vesicle with a smaller surface-to-volume ratio. In the case of our model system using granules, synexin and arachidonic acid, some type of expansion of the core matrix must have been sufficient to drive fusion, since the event was not dependent on MgATP or C1-. However, in the structured environment of the cytoplasm, expansion of the granules might be more difficult and require chemiosmotic forces. We cling to the view that such chemiosmotic swelling may be important in the living cell beause it is a view that has correctly predicted the behavior of seretory cells under certain circumstances. For example, secretion from cells can be blocked by increased osmotic strength of the culture medium, by pharmacological blockade of anion transport, or by proton ionophores (which short-circuit the proton pump), just as would be predicted if chemiosmotic swelling of granules were important in secretion [12,13].
17
Summary: conceptual integration of the model systems Fig. 1 presents a summary picture of the way in which the model system we have developed for membrane contact and fusion may fit into the overall mechanism of exocytosis in a cell. The figure also incorporates another model system for events before membrane fusion involving release of the granule from an actin-containing cytoskeleton [8], as explained in the legend. The overall scheme is a summation of individual steps that we have been able to recreate in the test tube. Are these steps in vitro artifacts, or do they really occur in the cell? This question poses the greatest challenge to us as model builders.
MODEL FOR PROCESS OF EXOCYTOSlS FROM CHROMAFFIN CELLS
co poun, Exocytosis[
Cytoskeleton
ctin,
\
~
Synexin,
(( isethionat~~FP,
)/ Pentalaminar Complex
/
///
,FP PMTHZ
Fig. l. Model for the process of exocytosis from chromaffin ceils. The events occurring during exocytosis are numbered in a temporal sequence, perhaps related to the increase in the free calcium concentration within the cell after some stimulus. Reaction 1 involves the Ca2+-dependent dissociation of the granule membrane from its cross-linked state with cytoskeletal actin, as the free calcium concentration rises above 10 -6 M. Thus, the secretory granule becomes specifically dislocated from its position in the cytoplasm. Subsequently, as the calcium concentration rises towards 10-5 M near the plasma membrane, reaction 2 becomes dominant and causes the secretory granule to become specifically attached to the inner aspect of the plasma membrane by means of Ca2~--activated synexin. This process is approximately equally sensitive to both the phenothiazine drugs trifluoperazine (TFP) and promethazine (PMTHZ) [6]. Following attachment, we presume that a granule-based osmotic gradient develops, possibly chemiosmotic in origin, and arachidonic acid release occurs. Both processes are represented by reaction 3, because at present we do not know the timing or the relative importance of the two. We presume that compound exocytosis can then occur, in which a free, intact granule can undergo reactions 2 and 3 with the granule membrane remnant of a past exocytotic event.
18
Acknowledgements Unpublished work described here on rabbit synexin was supported by Biomedical Research Support Grant 5 S07 RR05431-20 to the University of Virginia School of Medicine. We thank Shirley Davis for typing this manuscript.
References 1 Casey, R.P., Njus, O., Radda, G.K., and Sehr, P.A., ATP-evoked catecholamine release in chromaffin granules: osmotic lysis as a consequence of proton translation, Biochem. J., 158 (1976) 583-588. 2 Creutz, C.E., Cis-unsaturated fatty acids induce the fusion of chromaffin granules aggregated by synexin, J. Cell. Biol. 91 (1981) 247-256. 3 Creutz, C.E., Pazoles, C.J. and Pollard, H.B., Identification and purification of an adrenal medullary protein (synexin) that causes calcium dependent aggregation of isolated chromaffin granules, J. biol. Chem., 253 (1978) 2858-2866. 4 Creutz, C.E., Pazoles, C.J. and Pollard, H.B., Self-association of synexin in the presence of calcium, J. biol. Chem., 254 (1979) 553-558. 5 Creutz, C.E. and Pollard, H.B., A biophysical model of the chromaffin granule: accurate description of the kinetics of ATP and Cl--dependent granule lysis, Biophys. J. 31 (1980) 255 270. 6 Creutz, C.E., Scott, J.H., Pazoles, C.J. and Pollard, H.B., Further characterization of the aggregation and fusion of chromaffin granules by synexin as a model for compound exocytosis, J. cell. Biochem.. 18 (1982) 87-97. 7 Dabrow, M., Zaremba, S. and Hogue-Angeletti, R.A., Specificity of synexin-induced chromaffin granule aggregation, Biochem. Biophys. Res. Commun., 96 (1980) 1164-1171. 8 Fowler, V.M. and Pollard, H.B., Chromaffin granule membrane-F actin interactions are Ca 2+ sensitive, Nature (Lond.), 295 (1982) 336-339. 9 Hong, K., Duzgunes, N. and Papahadjopoulos, D., Role of synexin in membrane fusion, J. biol. Chem., 256 (1981) 3641-3644. 10 Morris, S.J. and Hughes J.M.X., Synexin protein is nonselective in its ability to increase Ca 2+dependent aggregation of biological and artifical membranes, Biochem. Biophys. Res. Commun., 91 (1979) 345-350. 11 Pazoles, C.J. and Pollard, H.B., Evidence for stimulation of anion transport in ATP-evoked transmitter release from isolated secretory vesicles, J. biol. Chem. 253 (1978) 3962-3969. 12 Pollard, H.B., Pazoles, C.J. and Creutz, C.E., Mechanism of calcium action and release of vesicle-bound hormones during exocytosis, Recent Progr. Horm. Res., 37 (1981) 299-332. 13 Pollard, H.B., Pazoles, C.J., Creutz, C.E. and Zinder, O., The chromaffin granule and possible mechanisms of exocytosis, Int. Rev. Cytol., 58 (1979) 160-198. 14 Scott, J.H., Creutz, C.E. and Pollard, H.B., Synexin binding to chromaffin cell plasma membrane. Europ. J. Cell. Biol., 22 (1980) 186 (Abstr.).