SNARE proteins and rab3A contribute to canalicular formation in parietal cells

SNARE proteins - rab3A - parietal cells - H+/K+-ATPase When stimulated by histamine, acetylcholine, or gastrin the luminal compartments of oxyntic parietal cells display conspicuous morphological changes. The luminal plasma membrane surface becomes greatly expanded, while the cytoplasmic tubulovesicles are decreased in parallel. Due to these membrane rearrangements the H+/K(+)-ATPase obtains access to the luminal surface, where proton secretion occurs. The stimulation-induced translocation of H+/K(+)-ATPase involves a fusion process. Exocytotic membrane fusion in neurons is achieved by the highly regulated interaction of mainly three proteins, the vesicle protein synaptobrevin and the plasma membrane proteins syntaxin and SNAP25 (synaptosomal-associated protein of 25 kDa), also referred to as SNARE proteins. Using immunofluorescence microscopy we analysed the subcellular distribution of neuronal synaptic proteins and rab3A in resting and stimulated parietal cells from pig and rat. In resting cells all synaptic proteins colocalized with the H+/ K(+)-ATPase trapped in the tubulovesicular compartment. After stimulation, translocated H+/K(+)-ATPase showed a typical canalicular distribution. Syntaxin, synaptobrevin, SNAP25 and rab3A underwent a similar redistribution in stimulated cells and consequently localized to the canalicular compartment. Using immunoprecipitation we found that the SNARE complex consisting of synaptobrevin, syntaxin and SNAP25, which is a prerequisite for membrane fusion in neurons, is also assembled in parietal cells. In addition the parietal cell-derived synaptobrevin could be proteolytically cleaved by tetanus toxin light chain. These data may provide evidence that SNARE proteins and rab3A are functionally involved in the stimulation-induced translocation of the H+/K(+)-ATPase.     

SNAP25, but not syntaxin 1A, recycles via an ARF6-regulated pathway in neuroendocrine cells

Soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins mediate cellular membrane fusion events and provide a level of specificity to donor-acceptor membrane interactions. However, the trafficking pathways by which individual SNARE proteins are targeted to specific membrane compartments are not well understood. In neuroendocrine cells, synaptosome-associated protein of 25 kDa (SNAP25) is localized to the plasma membrane where it functions in regulated secretory vesicle exocytosis, but it is also found on intracellular membranes. We identified a dynamic recycling pathway for SNAP25 in PC12 cells through which plasma membrane SNAP25 recycles in approximately 3 h. Approximately 20% of the SNAP25 resides in a perinuclear recycling endosome-trans-Golgi network (TGN) compartment from which it recycles back to the plasma membrane. SNAP25 internalization occurs by constitutive, dynamin-independent endocytosis that is distinct from the dynamin-dependent endocytosis that retrieves secretory vesicle constituents after exocytosis. Endocytosis of SNAP25 is regulated by ADP-ribosylation factor (ARF)6 (through phosphatidylinositol bisphosphate synthesis) and is dependent upon F-actin. SNAP25 endosomes, which exclude the plasma membrane SNARE syntaxin 1A, merge with those derived from clathrin-dependent endocytosis containing endosomal syntaxin 13. Our results characterize a robust ARF6-dependent internalization mechanism that maintains an intracellular pool of SNAP25, which is compatible with possible intracellular roles for SNAP25 in neuroendocrine cells.     

Loss of SNAP-25 and rabphilin 3a in sensory-motor cortex in Huntington's disease

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG-expansion in the gene encoding the protein huntingtin. The disease is characterized by progressive motor disturbances, cognitive defects, dementia, and weight loss. Using western blotting and immunohistochemistry we have assessed the expression levels and patterns of a number of proteins involved in neurotransmitter release in post-mortem frontal cortex samples from 10 HD cases with different disease grades. We report a loss of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein, synaptosome-associated protein 25 (SNAP 25) in HD brains of grades I-IV. Moreover, in brains of grade III and IV we found a reduction in rabphilin 3a, a protein involved in vesicle docking and recycling. These losses appear to be specific and not due to a general loss of synapses in the HD cortex. Thus, levels of synaptobrevin II, syntaxin 1, rab3a or synaptophysin are unaltered in the same patient samples. SNAP 25 and rabphilin 3a are crucial for neurotransmitter release. Therefore, we suggest that a deficient pre-synaptic transmitter release may underlie some of the symptoms of HD.     

The C terminus of SNAP25 is essential for Ca(2+)-dependent binding of synaptotagmin to SNARE complexes

The plasma membrane soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins syntaxin and synaptosome-associated protein of 25 kDa (SNAP25) and the vesicle SNARE protein vesicle-associated membrane protein (VAMP) are essential for a late Ca(2+)-dependent step in regulated exocytosis, but their precise roles and regulation by Ca(2+) are poorly understood. Botulinum neurotoxin (BoNT) E, a protease that cleaves SNAP25 at Arg(180)-Ile(181), completely inhibits this late step in PC12 cell membranes, whereas BoNT A, which cleaves SNAP25 at Gln(197)-Arg(198), is only partially inhibitory. The difference in toxin effectiveness was found to result from a reversal of BoNT A but not BoNT E inhibition by elevated Ca(2+) concentrations. BoNT A treatment essentially increased the Ca(2+) concentration required to activate exocytosis, which suggested a role for the C terminus of SNAP25 in the Ca(2+) regulation of exocytosis. Synaptotagmin, a proposed Ca(2+) sensor for exocytosis, was found to bind SNAP25 in a Ca(2+)-stimulated manner. Ca(2+)-dependent binding was abolished by BoNT E treatment, whereas BoNT A treatment increased the Ca(2+) concentration required for binding. The C terminus of SNAP25 was also essential for Ca(2+)-dependent synaptotagmin binding to SNAP25. syntaxin and SNAP25.syntaxin.VAMP SNARE complexes. These results clarify classical observations on the Ca(2+) reversal of BoNT A inhibition of neurosecretion, and they suggest that an essential role for the C terminus of SNAP25 in regulated exocytosis is to mediate Ca(2+)-dependent interactions between synaptotagmin and SNARE protein complexes.     

Role of SNAP23 in insulin-induced translocation of GLUT4 in 3T3-L1 adipocytes. Mediation of complex formation between syntaxin4 and VAMP2

Both syntaxin4 and VAMP2 are implicated in insulin regulation of glucose transporter-4 (GLUT4) trafficking in adipocytes as target (t) soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) and vesicle (v)-SNARE proteins, respectively, which mediate fusion of GLUT4-containing vesicles with the plasma membrane. Synaptosome-associated 23-kDa protein (SNAP23) is a widely expressed isoform of SNAP25, the principal t-SNARE of neuronal cells, and colocalizes with syntaxin4 in the plasma membrane of 3T3-L1 adipocytes. In the present study, two SNAP23 mutants, SNAP23-DeltaC8 (amino acids 1 to 202) and SNAP23-DeltaC49 (amino acids 1 to 161), were generated to determine whether SNAP23 is required for insulin-induced translocation of GLUT4 to the plasma membrane in 3T3-L1 adipocytes. Wild-type SNAP23 (SNAP23-WT) promoted the interaction between syntaxin4 and VAMP2 both in vitro and in vivo. Although SNAP23-DeltaC49 bound to neither syntaxin4 nor VAMP2, the SNAP23-DeltaC8 mutant bound to syntaxin4 but not to VAMP2. In addition, although SNAP23-DeltaC8 bound to syntaxin4, it did not mediate the interaction between syntaxin4 and VAMP2. Moreover, overexpression of SNAP23-DeltaC8 in 3T3-L1 adipocytes by adenovirus-mediated gene transfer inhibited insulin-induced translocation of GLUT4 but not that of GLUT1. In contrast, overexpression of neither SNAP23-WT nor SNAP23-DeltaC49 in 3T3-L1 adipocytes affected the translocation of GLUT4 or GLUT1. Together, these results demonstrate that SNAP23 contributes to insulin-dependent trafficking of GLUT4 to the plasma membrane in 3T3-L1 adipocytes by mediating the interaction between t-SNARE (syntaxin4) and v-SNARE (VAMP2).     

The heavy chain of conventional kinesin interacts with the SNARE proteins SNAP25 and SNAP23

Recent studies on the conventional motor protein kinesin have identified a putative cargo-binding domain (residues 827-906) within the heavy chain. To identify possible cargo proteins which bind to this kinesin domain, we employed a yeast two-hybrid assay. A human brain cDNA library was screened, using as bait residues 814-963 of human ubiquitous kinesin heavy chain. This screen initially identified synaptosome-associated protein of 25 kDa (SNAP25) as a kinesin-binding protein. Subsequently, synaptosome-associated protein of 23 kDa (SNAP23), the nonneuronal homologue of SNAP25, was also confirmed to interact with kinesin. The sites of interaction, determined from in vivo and in vitro assays, are the N-terminus of SNAP25 (residues 1-84) and the cargo-binding domain of kinesin heavy chain (residues 814-907). Both regions are composed almost entirely of heptad repeats, suggesting the interaction between heavy chain and SNAP25 is that of a coiled-coil. The observation that SNAP23 also binds to residues 814-907 of heavy chain would indicate that the minimal kinesin-binding domain of SNAP23 and SNAP25 is most likely residues 45-84 (SNAP25 numbering), a heptad-repeat region in both proteins. The major binding site for kinesin light chain in kinesin heavy chain was mapped to residues 789-813 at the C-terminal end of the heavy chain stalk domain. Weak binding of light chain was also detected at the N-terminus of the heavy chain tail domain (residues 814-854). In support of separate binding sites on heavy chain for light chain and SNAPs, a complex of heavy and light chains was observed to interact with SNAP25 and SNAP23.     

Interactions among the SNARE proteins and complexin analyzed by a yeast four-hybrid assay

Exocytosis is one of the most crucial and ubiquitous processes in all of biology. This event is mediated by the formation of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes, ternary assemblies of syntaxin, SNAP23/SNAP25 (synaptosomal-associated protein of 23 or 25 kDa), and synaptobrevin. The exocytotic process can be further regulated by complexin, which interacts with the SNARE complex. Complexin is involved in a Ca²⁺-triggered exocytotic process. In eukaryotic cells, multiple isoforms of SNARE proteins are expressed and are involved in distinct types of exocytosis. To understand the underlying biochemical mechanism of various exocytotic processes mediated by different SNARE protein isoforms, we systematically analyzed the interactions among syntaxin, SNAP23/SNAP25, synaptobrevin, and complexin by employing a newly developed yeast four-hybrid interaction assay. The efficiency of SNARE complex formation and the specificity of complexin binding are regulated by the different SNARE protein isoforms. Therefore, various types of exocytosis, occurring on different time scales with different efficiencies, can be explained by the involved SNARE complexes composed of different combinations of SNARE protein isoforms.     

Anaphase-promoting complex/cyclosome-Cdc-20 promotes Zwint-1 degradation

ZW10 interactor (Zwint-1) is an important component of the centromere and can recruit the dynamic protein kinase and dynein to promote chromosome movement and regulate the spindle assembly checkpoint (SAC). Zwint-1 activity is tightly regulated during the cell cycle. However, how the stability of Zwint-1 is regulated has not been clarified. Here, we show that the relative levels of Zwint-1 expression gradually decreased with the progression of cell cycling and decline sharply during mitotic exit. Treatment with cycloheximide reduced the levels of Zwint-1 while treatment with MG132 to inhibit endogenous ubiquitin-proteasome elevated the levels of Zwint-1 in HEK293T cells or Hela cells. Such data suggest that Zwint-1 may be degraded by endogenous ubiquitin-proteasome. Furthermore, induction of cell-division cycle protein 20 (Cdc20) overexpression decreased the levels of Zwint-1, which was abrogated by MG132 treatment. In contrast, Cdc20 silencing promoted the accumulation of Zwint-1. in vivo ubiquitination assay revealed that Cdc20 promoted the formation of Zwint-1 and ubiquitin-proteasome conjugates. Cotransfection with Cdc20 and wild-type Zwint-1, but not Zwint-1^ΔD-box, reduced the levels of Zwint-1. Immunoprecipitation and western blot analyses showed that Cdc20 interacted with wild-type Zwint-1, but not Zwint-1^ΔD-box although both Zwint-1 and Zwint-1^ΔD-box overexpression did not induce mitotic arrest. Collectively, our data indicated that Zwint-1 was ubiquitinated by anaphase-promoting complex/cyclosome (APC/C)-Cdc20 in a D-box-dependent manner. Therefore, the APC/C-Cdc20 controls the stability of Zwint-1, ensuring accurate regulation of the spindle assembly during the cell cycling in HEK293T cells.     

Identification of a novel SNAP25 interacting protein (SIP30)

Soluble N -ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), including synaptosome-associated proteins of 25 kDa (SNAP25), syntaxins, and vesicle-associated membrane proteins (VAMP), are essential for regulated exocytosis of synaptic vesicles in neurotransmission. We identified a cDNA coding for a novel protein of 266 amino acids that we have named SIP30 (S NAP25 interacting protein of 30 kDa). SIP30 is expressed abundantly in brain and slightly in testis and kidney. In brain, SIP30 is highly expressed in the inferior and superior colliculi, which contain important relay nuclei of the auditory and visual systems. GST-pull-down and immunoprecipitation assays showed direct binding of SIP30 to SNAP25. Although SIP30 does not directly interact with syntaxin based on pull-down assays, syntaxin does co-immunoprecipitate with SIP30 suggesting that syntaxin is indirectly associated with SIP30, perhaps through SNAP25.     

Rab3 reversibly recruits rabphilin to synaptic vesicles by a mechanism analogous to raf recruitment by ras.

GTP activates the interaction between the synaptic vesicle proteins rabphilin and rab3. This raises the question of whether rabphilin is a resident vesicle protein that recruits rab3 in a stage-dependent fashion, or if it is instead an effector protein recruited by rab3. We now show that rabphilin, like rab3, dissociates from synaptic vesicles after exocytosis in a manner requiring both Ca2+ and membrane fusion. Rabphilin interacts with GTP-rab3 via a N-terminal domain comprising a novel Zn2+(-)finger motif, and this interaction is essential for rabphilin binding to synaptic vesicles. Thus, in the same way that ras recruits raf to the plasma membrane, rab3 reversibly recruits rabphilin to synaptic vesicles in a stage-dependent manner. These results reveal an unexpected similarity between the molecular mechanisms by which small G protein function in recruiting effector proteins to membranes during membrane traffic and signal transduction.     

The Golgi matrix protein GM130: a specific interacting partner of the small GTPase rab1b

To detect specific partners of the small Golgi-localized GTPase rab1b we generated rab1b mutants and used them as bait proteins in yeast two-hybrid screens. We isolated several specifically interacting clones. Two of them encode large protein fragments highly homologous to rat GM130 and to human Golgin95. The full-length human GM130 cDNA was cloned and its interaction with rab1b was characterized in detail by yeast two-hybrid and in vitro binding assays. Here we report for the first time that the rab1b protein interacts specificially with GM130 in a GTP-dependent manner and therefore needs the hypervariable regions of the N- and C-termini. We mapped the rab1b binding site of GM130 and provide evidence that it is different to the previously described p115 and Grasp65 binding sites of the GM130 protein.     

Negative Regulation of Syntaxin4/SNAP-23/VAMP2-Mediated Membrane Fusion by Munc18c In Vitro

Background

Translocation of the facilitative glucose transporter GLUT4 from an intracellular store to the plasma membrane is responsible for the increased rate of glucose transport into fat and muscle cells in response to insulin. This represents a specialised form of regulated membrane trafficking. Intracellular membrane traffic is subject to multiple levels of regulation by conserved families of proteins in all eukaryotic cells. Notably, all intracellular fusion events require SNARE proteins and Sec1p/Munc18 family members. Fusion of GLUT4-containing vesicles with the plasma membrane of insulin-sensitive cells involves the SM protein Munc18c, and is regulated by the formation of syntaxin 4/SNAP23/VAMP2 SNARE complexes.

Methodology/Principal Findings

Here we have used biochemical approaches to characterise the interaction(s) of Munc18c with its cognate SNARE proteins and to examine the role of Munc18c in regulating liposome fusion catalysed by syntaxin 4/SNAP23/VAMP2 SNARE complex formation. We demonstrate that Munc18c makes contacts with both t- and v-SNARE proteins of this complex, and directly inhibits bilayer fusion mediated by the syntaxin 4/SNAP23/VAMP2 SNARE complex.

Conclusion/Significance

Our reductionist approach has enabled us to ascertain a direct inhibitory role for Munc18c in regulating membrane fusion mediated by syntaxin 4/SNAP23/VAMP2 SNARE complex formation. It is important to note that two different SM proteins have recently been shown to stimulate liposome fusion mediated by their cognate SNARE complexes. Given the structural similarities between SM proteins, it seems unlikely that different members of this family perform opposing regulatory functions. Hence, our findings indicate that Munc18c requires a further level of regulation in order to stimulate SNARE-mediated membrane fusion.     

Membrane targeting and activation of the Lowe syndrome protein OCRL1 by rab GTPases

The X-linked disorder oculocerebrorenal syndrome of Lowe is caused by mutation of the OCRL1 protein, an inositol polyphosphate 5-phosphatase. OCRL1 is localised to the Golgi apparatus and early endosomes, and can translocate to lamellipodia upon growth factor stimulation. We show here that OCRL1 interacts with several members of the rab family of small GTPases. Strongest interaction is seen with Golgi-associated rab1 and rab6 and endosomal rab5. Point mutants defective in rab binding fail to target to the Golgi apparatus and endosomes, strongly suggesting rab interaction is required for targeting of OCRL1 to these compartments. Membrane recruitment via rab binding is required for changes in Golgi and endosomal dynamics induced by overexpression of catalytically inactive OCRL1. In vitro experiments demonstrate that rab5 and rab6 directly stimulate the 5-phosphatase activity of OCRL1. We conclude that rabs play a dual role in regulation of OCRL1, firstly targeting it to the Golgi apparatus and endosomes, and secondly, directly stimulating the 5-phosphatase activity of OCRL1 after membrane recruitment.     

The ras-like protein p25rab3A is partially cytosolic and is expressed only in neural tissue.

An antipeptide antiserum has been developed against a sequence near the C terminus of the small guanine nucleotide-binding protein p25rab3A. This protein is the product of one of a large number of genes that show homology to the ras proto-oncogenes. Immunoblotting with the antiserum specifically detected a 25-kilodalton protein in brain membranes. This protein coeluted from a MonoQ high-resolution ion-exchange column with a 25-kilodalton GTP-binding protein at a salt concentration similar to that known to elute purified p25rab3A. Unlike p21ras, which is exclusively membrane bound, p25rab3A is present in both the cytosol and membrane fractions of rat brain. It was not detected in other tissues, although a band of slightly lower molecular weight was observed with skeletal muscle. Western blot (immunoblot) analysis of five regions of the rat brain indicated that p25rab3A is most abundant in the hypothalamus and hippocampus.     

EBAG9 adds a new layer of control on large dense-core vesicle exocytosis via interaction with Snapin

Regulated exocytosis is subject to several modulatory steps that include phosphorylation events and transient protein-protein interactions. The estrogen receptor-binding fragment-associated gene9 (EBAG9) gene product was recently identified as a modulator of tumor-associated O-linked glycan expression in nonneuronal cells; however, this molecule is expressed physiologically in essentially all mammalian tissues. Particular interest has developed toward this molecule because in some human tumor entities high expression levels correlated with clinical prognosis. To gain insight into the cellular function of EBAG9, we scored for interaction partners by using the yeast two-hybrid system. Here, we demonstrate that EBAG9 interacts with Snapin, which is likely to be a modulator of Synaptotagmin-associated regulated exocytosis. Strengthening of this interaction inhibited regulated secretion of neuropeptide Y from PC12 cells, whereas evoked neurotransmitter release from hippocampal neurons remained unaltered. Mechanistically, EBAG9 decreased phosphorylation of Snapin; subsequently, association of Snapin with synaptosome-associated protein of 25 kDa (SNAP25) and SNAP23 was diminished. We suggest that the occurrence of SNAP23, Snapin, and EBAG9 also in nonneuronal cells might extend the modulatory role of EBAG9 to a broad range of secretory cells. The conjunction between EBAG9 and Snapin adds an additional layer of control on exocytosis processes; in addition, mechanistic evidence is provided that inhibition of phosphorylation has a regulatory function in exocytosis.     

ATP dependence of the SNARE/caveolin 1 interaction in the hippocampus

The molecular mechanisms underlying the regulation of neurotransmission has been an open question for many years. Here, we have examined an interaction between caveolin1 and SNAREs (soluble N-ethylmalemide-sensitive factor attachment protein receptor) which may contribute to the cellular mechanisms underlying changes in synaptic strength. Previously, we reported that application of 4-aminopyridine to hippocampal slices resulted in a persistent potentiation of synaptic transmission and the induction of a short-lasting and specific 40-kDa complex composed of synaptosomal associated protein of 25 kDa (SNAP25) and caveolin1. We have characterized the binding properties of these proteins and observed that in vitro caveolin1 directly associates with both SNAP25 and syntaxin. Caveolin/SNARE interactions are enhanced in the presence of ATP by a mechanism that involves phosphorylation. While caveolin has been associated with cholesterol transport, signal transduction, and transcytosis, this study provides evidence that caveolin is also a SNARE accessory protein.     

A gain-of-function mutant of Munc18-1 stimulates secretory granule recruitment and exocytosis and reveals a direct interaction of Munc18-1 with Rab3

Munc18-1 plays a crucial role in regulated exocytosis in neurons and neuroendocrine cells through modulation of vesicle docking and membrane fusion. The molecular basis for Munc18 function is still unclear, as are the links with Rabs and SNARE [SNAP (soluble N-ethylmaleimide-sensitive factor-attachment protein) receptor] proteins that are also required. Munc18-1 can bind to SNAREs through at least three modes of interaction, including binding to the closed conformation of syntaxin 1. Using a gain-of-function mutant of Munc18-1 (E466K), which is based on a mutation in the related yeast protein Sly1p, we have identified a direct interaction of Munc18-1 with Rab3A, which is increased by the mutation. Expression of Munc18-1 with the E466K mutation increased exocytosis in adrenal chromaffin cells and PC12 cells (pheochromocytoma cells) and was found to increase the density of secretory granules at the periphery of PC12 cells, suggesting a stimulatory effect on granule recruitment through docking or tethering. Both the increase in exocytosis and changes in granule distribution appear to require Munc18-1 E466K binding to the closed form of syntaxin 1, suggesting a role for this interaction in bridging Rab- and SNARE-mediated events in exocytosis.     

Carboxyl-methylation of Rab3D in the rat pancreatic acinar tumor cell line AR42J.

Rab3D is a small GTPase implicated in regulated exocytosis, and is a marker of secretory granules in exocrine cells. We have previously shown that rab3D undergoes reversible carboxyl-methylation in adult rat pancreatic acinar cells, and that carboxyl-methylation of rab3D is developmentally regulated concomitantly with the maturation of the regulated secretory apparatus in rat pancreas. We also observed that dexamethasone treatment of the rat pancreatic acinar tumor cell line, AR42J, led to a significant increase in the size of the unmethylated pool of a rab3-like protein. The current study was designed to further characterize this rab3-like protein. Here we show that AR42J cells express rab3D, and that the protein focuses on 2D gels as two spots with pI values of 4.9 and 5.0. Treatment of AR42J cells with N-acetyl-S-geranylgeranyl-l-cysteine, an inhibitor of carboxyl-methylation, led to a decrease in the basic form of rab3D and a proportional increase in the acidic form. In contrast, N-acetyl-S-farnesyl-l-cysteine, which inhibits carboxyl-methylation of farnesylated proteins, had no effect. Lovastatin, an inhibitor of geranylgeranylation, also induced an accumulation of the acidic form of rab3D. Taken together, these data indicate that rab3D can undergo reversible carboxyl-methylation in AR42J cells by a geranylgeranyl-specific methyltransferase. The 2D gel and immunoblotting analyses indicated that dexamethasone treatment of AR42J cells led to an increase in the proportion of the unmethylated form of rab3D concurrent to inducing a regulated secretory pathway, similar to the rab3D profile change in developing rat pancreas. Our data, along with previous studies done on developing rat pancreas, indicate that the tumor cell line AR42J represents a good model system for studying the regulated secretory pathway, and that carboxyl-methylation of rab3D may play a role in the acquisition of stimulus-secretion coupling.