GTP-dependent Ca2+ release from rat liver microsomes. Vesicle fusion is not required

The GTP-dependent calcium release from rat liver microsomes is known to be promoted in the presence of colloids like polyethyleneglycol (PEG), polyvinylpyrrolidine, or albumin. Dawson et al. [(1987) Biochem. J. 244, 87-92] using the 'fusogen' PEG have concluded that both GTP-induced calcium efflux and the enhancement of InsP3-promoted calcium release in the presence of GTP could be attributed to a GTP-dependent vesicle fusion. Here, using the more physiological colloid albumin we report that GTP-induced calcium release from rat liver microsomes may not be linked to vesicle fusion.     

Liver cytosolic non-dialysable factor(s) can counteract GTP-dependent Ca2+ release in rat liver microsomal fractions

Readdition to rat liver microsomes of dialysed liver post-microsomal supernatant resulted in an almost complete inhibition of the Ca2+-releasing effect of GTP. Such inhibition was heat-labile, and was associated with non-ultrafiltrable supernatant components with a molecular weight higher than 30,000 D. A preliminary fractionation of liver supernatant showed that the inhibitory effect is recovered in the 40-50% ammonium sulfate-precipitated proteins, with an approx. 10-fold enrichment. The active ammonium sulfate fraction did not modify the GTP-induced Ca2+ increase of passive Ca2+ efflux from microsomes, nor did it affect microsomal GTP hydrolysis, which is likely required for its Ca2+ releasing effect. The active ammonium sulfate fraction appears to markedly favour the translocation of GTP-released Ca2+ into a microsomal GTP-insensitive pool. Separation of liver microsomes in smooth and rough fractions revealed that such GTP-insensitive Ca2+ pool is almost completely associated with smooth microsomes.     

The mechanism of action of GTP on Ca2+ efflux from rat liver microsomal vesicles.

1. Guanosine 5'-[gamma-thio]triphosphate (GTP[S]), if added before GTP, blocks both Ca2+ efflux promoted by GTP and the effect of GTP on enhancement of inositol 1,4,5-triphosphate (IP3)-promoted Ca2+ release from preloaded microsomal vesicles. If, however, GTP[S] is added after GTP, it does not reverse the Ca2+ efflux promoted by GTP, nor does it inhibit IP3-promoted Ca2+ release. 2. The effect of GTP in enhancing IP3-promoted Ca2+ release is maintained after washing the microsomal vesicles free of added GTP. After this treatment, enhancement of IP3-promoted Ca2+ efflux can be observed in the absence of poly(ethylene glycol). 3. Electron microscopy shows that during GTP treatment of microsomal vesicles there is rapid production of very large vesicular structures, apparently produced by fusion of smaller vesicles. 4. Light-scattering changes are detectable during the fusion process. 5. Both Ca2+ efflux promoted by GTP and the enhancement of IP3-promoted Ca2+ release seen in the presence of GTP can probably be attributed to GTP-dependent vesicle fusion.     

Evidence for a GTP-dependent increase in membrane permeability for calcium in NG108-15 microsomes

The effect of GTP on Ca2+ uptake and release was studied in a microsomal fraction isolated from neuroblastoma x glioma hybrid NG108-15 cells. GTP did not alter the ATP-dependent initial uptake of Ca2+ but markedly enhanced the efflux of Ca2+ from microsomes. GTP-dependent Ca2+ release requires the presence of millimolar concentration of Mg2+. The effect of GTP was not mimicked by other nucleotides and was competitively blocked by the thiophosphate analogue of GTP, GTP gamma S but not by the non-hydrolyzable nucleotide GMP-PNP. Addition of an inhibiting concentration of GTP gamma S after completion of GTP-induced calcium release did not result in a re-uptake of Ca2+, showing the irreversibility of the releasing effect of GTP. Our data are consistent with the hypothesis of Ca2+-dependent GTP-induced opening of a channel responsible for vectorial transport of Ca2+ ions from one intracellular compartment to another. A model is proposed suggesting that the GTP-binding protein is a GTP-specific diacylglycerol kinase.     

GTP-dependent membrane fusion during hepatocarcinogenesis and liver regeneration

Rough microsomes were isolated from homogenates of livers of rats bearing hepatomas as well as from homogenates of livers of rats 24 and 48 h after partial hepatectomy. When incubated in the presence of GTP in a cell-free system to assay membrane fusion these membranes were observed to have a greater capacity (1.4 to 5 fold) for GTP-dependent fusion than homologous membranes from control non-proliferating liver tissue. The enhanced GTP-dependent membrane fusion may reflect changes in membrane properties related to cell proliferation.     

Effect of specific proteolytic cleavages on tubulin polymer formation.

The capacity for self-polymerization and shape of the tubulin polymers assembled after digestion with trypsin, Pronase, chymotrypsin, subtilisin, Staphylococcus aureus proteinase V8 and proteinase K were investigated. Digestion with trypsin, Pronase or chymotrypsin resulted in a decrease in the ability of tubulin for self-assembly, whereas limited proteolysis with subtilisin, S. aureus proteinase V8 or proteinase K resulted in an increase in such ability. The shape of the assembled polymers varied from typical microtubules (after the treatment with trypsin or Pronase) to sheets (after the treatment with chymotrypsin) and from hooked microtubules with a constant polarity (after the treatment with subtilisin) to the disappearance of a defined polarity of such polymers (after the treatment with S. aureus V8 proteinase or proteinase K). These results indicate that the tubulin C-terminal regions are involved in the regulation of microtubule polymerization, shape, directional growth and lateral interactions between tubulin protofilaments.     

GTP-mediated Ca2+ release in rough endoplasmic reticulum. Correlation with a GTP-sensitive increase in membrane permeability.

Guanine nucleotides have been reported to stimulate reticular Ca2+ release. By using the structure-linked latency of microsomal mannose-6-phosphate phosphatase as an index of microsomal permeability [Arion, Ballas, Lange & Wallin (1976) J. Biol. Chem. 251, 4901-4907], the effects of GTP on Ca2+ release and membrane permeability were compared in liver microsomes. In a stripped rough-microsome preparation, GTP caused a dose-dependent increase in mannose 6-phosphate permeability. Half-maximal and maximal effects were observed at 3 microM- and 10 microM-GTP respectively. The time course of the change in membrane permeability coincided with the time course of GTP-dependent Ca2+ release. This increase in microsomal permeability displayed positive to-operativity with respect to GTP (Hill coefficient = 1.8). By analogy to the GTP-dependent Ca2+ release process, guanosine 5'-[gamma-thio]triphosphate and guanosine 5'-[beta gamma-imido]-triphosphate inhibited the ability of GTP to alter microsomal permeability, but were without effect when added alone. In the presence of 50 microM-GTP, complete inhibition of the GTP-dependent increase in microsomal permeability was achieved with 10 microM-guanosine 5'-[gamma-thio]triphosphate, whereas a 25% inhibition was observed with 10 microM-guanosine 5'-[beta gamma-imido]triphosphate. In contrast with previous observations in crude microsomal preparations, GTP-dependent Ca2+ release in the stripped rough-microsome preparation did not require the addition of poly(ethylene glycol), although the latter did stimulate the rate of Ca2+ release. The ability of GTP to alter microsomal permeability was blocked by prior treatment with the thiol reagent p-hydroxymercuribenzoate; complete inhibition was observed after a 10 min exposure to 50 microM. Inhibition was reversed by subsequent treatment with dithiothreitol. The marked similarities between the two GTP-sensitive processes indicate that they may function via the same mechanism.     

Primary structure and possible origin of the non-glycosylated basic proline-rich protein of human submandibular/sublingual saliva.

As a first step in determining the molecular mechanism of membrane fusion stimulated by GTP in rough endoplasmic reticulum (RER), we have looked for GTP-binding proteins. Rough microsomes from rat liver were treated for the release of ribosomes, and the membrane proteins were separated by SDS/polyacrylamide-gel electrophoresis. The polypeptides were then blotted on to nitrocellulose sheets and incubated with [alpha-32P]GTP [Bhullar & Haslam (1987) Biochem. J. 245, 617-620]. A doublet of polypeptides (23 and 24 kDa) was detected in the presence of 2 microM-MgCl2. Binding of [alpha-32P]GTP was blocked by 1-5 mM-EDTA, 10-10,000 nM-GTP or 10 microM-GDP. Either guanosine 5'-[gamma-thio]triphosphate or guanosine 5'-[beta gamma-imido]triphosphate at 100 nM completely inhibited binding, but ATP, CTP or UTP at 10 mciroM did not. Pretreatment of microsomes by mild trypsin treatment (0.5-10 micrograms of trypsin/ml, concentrations known not to affect microsomal permeability) led to inhibition of [alpha-32P]GTP binding, suggesting a cytosolic membrane orientation for the GTP-binding proteins. Two-dimensional gel-electrophoretic analysis revealed the 23 and 24 kDa [alpha-32P]GTP-binding proteins to have similar acid isoelectric points. [alpha-32P]GTP binding occurred to similar proteins of rough microsomes from rat liver, rat prostate and dog pancreas, as well as to a 23 kDa protein of rough microsomes from frog liver, but occurred to distinctly different proteins in a rat liver plasma-membrane-enriched fraction. Thus [alpha-32P]GTP binding has been demonstrated to two low-molecular-mass (approx. 21 kDa) proteins in the rough endoplasmic reticulum of several varied cell types.     

Polyethylene glycol-stimulated microsomal GTP hydrolysis. Relationship to GTP-mediated Ca2+ release

It has recently been observed that GTP mediates Ca2+ release from internal Ca2+ stores. In contrast to effects on permeabilized cells, GTP-dependent Ca2+ release in isolated microsomes requires the presence of polyethylene glycol (PEG). We have investigated the effects of PEG on microsomal GTPase activity and report that PEG stimulates a high-affinity (Km = 0.9 microM) GTPase. The effects of PEG reflect an increase in the Vmax of this activity; no effects on Km were observed. The concentration dependence for PEG-dependent stimulation of the high-affinity GTPase exactly mimicked that for GTP-dependent Ca2+ release. The stimulation of GTP hydrolysis by PEG was specific for the microsome fraction; only small effects were obtained with plasma membrane or cytosol fractions. As observed for GTP-dependent Ca2+ release, the microsomal PEG-stimulated GTPase was competitively inhibited by the GTP analog GTP gamma S (Ki = 60 nM). It is proposed that the PEG-stimulated GTPase may represent an intrinsic activity of the guanine nucleotide binding protein involved in the regulation of reticular Ca2+ fluxes.     

The membrane of the rough endoplasmic reticulum contains cytoplasmically exposed high affinity GTP-binding sites

Binding experiments using [14C]GTP and rat liver rough microsomes gave Scatchard plots with Kd congruent to 0.1 microM and a binding capacity congruent to 40 pmol/mg microsomal protein. Removal of the ribosomes did not modify the binding parameters. GTP was competed out by GTP analogues but not by ATP. Limited proteolysis of rough microsomes decreased the GTP-binding capacity and prevented GTP from suppressing the latency of mannose-6-phosphatase and of the synthesis of dolichol-linked chitobiose. The GTP-binding protein is probably involved in these effects of GTP. Its function could be to act in the association-dissociation of membrane components at the ribosome-membrane junction.     

Protease-activated protein kinase in rat liver plasma membrane

Upon limited proteolysis with trypsin, a cAMP and Ca2+-independent protein kinase was produced from rat liver plasma membrane. This enzyme showed a multifunctional capacity and phosphorylated calf thymus histone and rat liver ribosomal proteins. The molecular weight was estimated to be 5.0 X 10(4). When plasma membrane was treated with a buffer containing Triton X-100, a proenzyme with a molecular weight of 8.4 X 10(4) was extracted. By tryptic digestion, the proenzyme was converted to an active protein kinase which was similar to the enzyme obtained by the direct digestion of membrane. However, this proenzyme phosphorylated H1 histone in the presence of Ca2+ and phospholipid without proteolytic digestion. These results indicate the existence of a protease-activated protein kinase in rat liver plasma membrane and the proenzyme seems to be same as protein kinase C.     

Characteristics of GTP-mediated microsomal Ca2+ release

Guanosine triphosphate (GTP) can release Ca2+ and enhance responses to D-myo-inositol 1,4,5-trisphosphate (IP3) in crude liver microsomes in the presence of polyethylene glycol (PEG) (Dawson et al. (1986) Biochem. J. 234, 311-315). The mechanism of these responses has been further investigated. GTP gamma S which antagonizes the actions of GTP on microsomes, does not promote Ca2+ re-uptake when added after the completion of GTP-mediated Ca2+ release. However, the effects of GTP could be reversed by washing or dilution of the microsomes. Addition of PEG to the incubation medium promoted the aggregation of microsomes. Electron microscopy provided no evidence for the fusion of microsomal vesicles in the presence or absence of GTP. In the presence of PEG, GTP produced an alteration of the permeability properties of the microsomal membrane as indicated by increased leakage of an intraluminal esterase, a reduction in the mean buoyant density of the vesicles, and a decrease in the latency of mannose 6-phosphate hydrolysis. All three effects developed relatively slowly, whereas the effects of GTP on Ca2+ fluxes occurred more rapidly (complete within 15 min). A low permeability to mannose 6-phosphate was restored upon washing away the GTP. These results suggest that non-specific permeability changes may underly the effects of GTP on Ca2+ release and that, under certain conditions, GTP can reversibly modulate the permeability of a transmembrane 'pore' in microsomal membranes that can pass ions and macromolecules. The possibility that such a pore serves to link IP3-sensitive vesicles with other Ca2+-containing compartments is discussed.     

Acidic pH generated by H+-ATPase pumps triggers the activity of a fusogenic protein associated with rat liver endoplasmic reticulum.

Fusogenic protein (FP) is a glycoprotein ( approximately 50 kDa), previously purified by us from rat liver endoplasmic reticulum, which explicates fusogenic activity at acidic pH in vitro. To suggest a possible role of FP in membrane fusion, the topology of the protein in the membrane and the conditions in which FP is operating in microsomes have been investigated. Anti-FP polyclonal antibodies inhibited pure FP activity, but not the protein activity in microsomes, suggesting interaction of antibodies with a part of FP concealed in intact membranes. FP activity in microsomes was lost after treatment with Pronase. Western blot analysis of Pronase-treated microsomes showed that the proteolysis removed a fragment ( approximately 5 kDa). This fragment is exposed on the outer surface of microsomes and involved in fusogenic activity, whereas the largest part of FP is embedded in microsomal vesicles. Therefore, FP can be affected by modifications on the cytosolic and luminal sides of microsomal membranes. Indeed, when microsomal lumen was acidified by H+-ATPase activity, binding and fusion of fluorescent labelled liposomes to microsomes occurred. Direct involvement of FP in the fusogenic event was observed by reconstituting pure FP in liposomes with a preformed H+ gradient. FP triggered a fusion process in response to the acidic interior of liposomes, despite an exterior 7.4 pH unable to promote fusogenic protein activity. As intracellular membrane fusion occurs at neutral pH involving the cytosolic sides of membranes, FP may participate in this event by exploiting the acidic pH formed in the lumen of endoplasmic reticulum through H+-translocating ATPase activity.     

MgATP-dependent glucose 6-phosphate-stimulated Ca2+ accumulation in liver microsomal fractions. Effects of inositol 1,4,5-trisphosphate and GTP

Ca2+ release triggered by inositol 1,4,5-trisphosphate (IP3) and/or GTP has been studied with rough and smooth microsomes isolated from rat liver. Microsomes were loaded with Ca2+ in the presence of MgATP and in the presence or in the absence of glucose 6-phosphate (glucose-6-P) which markedly stimulated the MgATP-dependent Ca2+ accumulation in rough and smooth microsomes (5- and 10-fold, respectively). Upon addition of IP3 (5 microM), rough and smooth microsomes rapidly release a part (not exceeding 20%) of the Ca2+ previously accumulated both in the absence and in the presence of glucose-6-P. Under the same experimental conditions, inositol 1,3,4,5-tetrakisphosphate was ineffective in triggering any Ca2+ release. Upon addition of GTP (10 microM) both the microsomal fractions progressively release the Ca2+ previously accumulated in the presence of glucose-6-P, when 3% polyethylene glycol was also present. In the absence of polyethylene glycol, GTP released Ca2+ from rough microsomes only, and GTP plus IP3 caused a Ca2+ release which was the sum of the Ca2+ releases caused by GTP and IP3 independently. Both IP3 and GTP, added to microsomes at the beginning of the glucose-6-P-stimulated Ca2+ uptake, reduced the Ca2+ accumulation into rough and smooth microsomes without modifying the initial rate (3 min) of Ca2+ uptake. Also in these conditions, the effects of GTP and IP3 were merely additive. These results indicate that both rough and smooth liver microsomes are responsive to IP3 and GTP with respect to Ca2+ release and that IP3 and GTP likely act independently.     

Partial truncation of the NH2-terminus affects physical characteristics and membrane binding, aggregation, and fusion properties of annexin A7

Annexin A7 (synexin, annexin VII), a member of the annexin family of proteins, causes aggregation of membranes in a Ca2+-dependent manner and has been suggested to promote membrane fusion during exocytosis of lung surfactant, catecholamines, and insulin. Although annexin A7 (A7) was one of the first annexin proteins described, limited studies of its physical characteristics or of structural domains affecting any of its proposed functions have been conducted. As postulated for other annexin proteins, the unique NH2-domain possibly determines the functional specificity of A7. Therefore, we evaluated the effects of segmental deletions in the NH2-terminus on several characteristics associated with the COOH-terminus of A7. The COOH-terminus contains the only tryptophan residue, and all potential trypsin sites, and the Ca2+ and phospholipid binding sites. Recombinant rat A7 and its deletion mutants were expressed using constructs based on the cDNA sequence obtained by screening a rat lung cDNA library. Ca2+ increased the tryptophan fluorescence of A7 and caused a small red shift in the emission maximum (lambdamax), which was further increased in presence of phospholipid vesicles (PLV). NH2-terminal deletions of 29, 51, and 109 residues affected the peak width of fluorescence and lambdamax, surface-exposure of tryptophan residue, and caused a smaller Ca2+-dependent red shift in lambdamax of membrane-bound protein in comparison to A7. Limited proteolysis with trypsin showed that Ca2+ increased the proteolysis of all proteins, but the deletions also affected the pattern of proteolysis. The presence of PLV protected against Ca2+-dependent increase in proteolysis of all proteins. The deletion of first 29 residues also caused decreased membrane binding, aggregation, and fusion, when compared with A7. Collectively, these results suggest that specific NH2-terminus domains can alter those properties of A7 that are normally associated with the COOH-terminus. We speculate that interactions between the NH2- and COOH-termini are required for membrane binding, and aggregation and fusion properties of annexin A7.     

Effects of GTP on Ca2+ movements across endoplasmic reticulum membranes

Our initial observation that GTP could, under some experimental conditions, have profound effects on Ca2+ movements across endoplasmic reticulum membranes arose from attempts to increase the sensitivity of rat liver microsomes to inositol 1,4,5 trisphosphate (IP3). Most preparations of microsomal fractions from rat liver release only a very small percentage of accumulated Ca2+ on addition of IP3. We found, rather empirically, that the addition of microM concentrations of GTP greatly enhanced the amount of Ca2+ releasable by IP3. The initial, very appealing, hypothesis was to postulate a direct effect of GTP on the IP3-sensitive Ca2+ channel. This idea is no longer tenable, as will be described below. The more likely explanation, that GTP has its effect by either fusing small microsomal vesicles together or by allowing some form of communication between adjacent membranes is considerably more complex mechanistically and also possibly has far reaching implications for the mechanisms by which cells organise and maintain their reticular structures.     

GTP-dependent regulation of myometrial KCa channels incorporated into lipid bilayers

The regulation of calcium-activated K (KCa) channels by a G protein-mediated mechanism was studied. KCa channels were reconstituted in planar lipid bilayers by fusion of membrane vesicles from rat or pig myometrium. The regulatory process was studied by exploring the actions of GTP and GTP gamma S on single channel activity. KCa channels had a conductance of 260 +/- 6 pS (n = 25, +/- SE, 250/50 mM KCl gradient) and were voltage dependent. The open probability (Po) vs. voltage relationships were well fit by a Boltzmann distribution. The slope factor (11 mV) was insensitive to internal Ca2+. The half activation potential (V1/2) was shifted -70 mV by raising internal Ca2+ from pCa 6.2 to pCa 4. Addition of GTP or GTP gamma S activated channel activity only in the presence of Mg2+, a characteristic typical of G protein-mediated mechanisms. The Po increased from 0.18 +/- 0.08 to 0.49 +/- 0.07 (n = 7, 0 mV, pCa 6 to 6.8). The channel was also activated (Po increased from 0.03 to 0.37) in the presence of AMP-PNP, a nonphosphorylating ATP analogue, suggesting a direct G protein gating of KCa channels. Upon nucleotide activation, mean open time increased by a factor of 2.7 +/- 0.7 and mean closed time decreased by 0.2 +/- 0.07 of their initial values (n = 6). Norepinephrine (NE) or isoproterenol potentiated the GTP-mediated activation of KCa channels (Po increased from 0.17 +/- 0.06 to 0.35 +/- 0.07, n = 10). These results suggest that myometrium possesses beta-adrenergic receptors coupled to a GTP-dependent protein that can directly gate KCa channels. Furthermore, KCa channels, beta-adrenergic receptors, and G proteins can be reconstituted in lipid bilayers as a stable, functionally coupled, molecular complex.     

cAMP-dependent phosphorylation of RER proteins from rat liver: relationship with GTP-dependent membrane fusion.

Incubation of stripped rough microsomes (SRM) with the catalytic subunit of protein kinase A (PKA) permitted specific phosphorylation of seven proteins having relative molecular mass values of 55, 35, 23, 22.5, 22, 18.5 and 16.5 kDa (P55, P35 etc.). By two dimensional gel analysis, we compared these phosphoproteins with low-molecular-weight GTP-binding proteins and revealed that P23 and P22.5 co-migrated with known GTP-binding proteins. Next we examined the effect of cAMP-dependent phosphorylation on a GTP-dependent membrane function, membrane fusion. Quantitative analysis indicated no difference in the amount of membrane fusion obtained whether SRM were incubated in the absence or in the presence of PKA. Thus several rough microsomal proteins underwent cAMP-dependent phosphorylation and this post-translational modification did not affect GTP-dependent membrane fusion in a cell free system.     

GTP-dependent permeabilized neutrophil secretion requires a freely diffusible cytosolic protein

Guanosine triphosphate (GTP) has been implicated in the regulation of Ca(2+)-mediated secretion from neutrophils. We further examined the role of GTP in neutrophil secretion using streptolysin O permeabilized cells. We found that, in the presence of GTP, 1.0 microM free Ca(2+) causes maximum secretion-equivalent to that achieved with 100 microM free Ca(2+)-whereas GTPgammaS inhibits Ca(2+)-stimulated secretion. Interestingly, GTP by itself stimulates secretion. These results indicate the existence of a GTP-regulated mechanism of secretion in neutrophils that requires GTP hydrolysis to stimulate secretion in the presence and absence of Ca(2+). The stimulatory effect of GTP is only observed when GTP is present during permeabilization. Addition of GTP after permeabilization, when the cytosolic contents have leaked out from cells, gives no stimulatory response, implying that the GTP-dependent secretory apparatus requires at least one cytosolic protein. GTP-dependent secretion can be reconstituted with crude HL-60 and bovine liver cytosol. The reconstituting activity binds to GTP-agarose, suggesting that the cytosolic factor is a GTP-binding protein or forms a complex with a GTP-binding protein. However, it is not a member of the rho or rac families of GTPases. By gel filtration chromatography, the secretion-reconstituting activity eluted at 870 and 200 kDa, but in the presence of GTP, eluted at 120 kDa, indicating that it is part of a high-molecular-weight complex that dissociates in the presence of GTP. Retention of adenosine diphosphate-ribosylation factor (ARF) in permeabilized cells and insensitivity of the cytosolic reconstituting activity to brefeldin A led to our speculation that ARF6 may be the GTPase involved in GTP-dependent secretion, and that activity from a BFA-insensitive ARF6 guanine nucleotide exchange factor reconstitutes secretion.     

RalA-exocyst interaction mediates GTP-dependent exocytosis

Many secretory cells utilize a GTP-dependent pathway, in addition to the well characterized Ca2+-dependent pathway, to trigger exocytotic secretion. However, little is currently known about the mechanism by which this may occur. Here we show the key signaling pathway that mediates GTP-dependent exocytosis. Incubation of permeabilized PC12 cells with soluble RalA GTPase, but not RhoA or Rab3A GTPases, strongly inhibited GTP-dependent exocytosis. A Ral-binding fragment from Sec5, a component of the exocyst complex, showed a similar inhibition. Point mutations in both RalA (RalA(E38R)) and the Sec5 (Sec5(T11A)) fragment, which abolish RalA-Sec5 interaction also abolished the inhibition of GTP-dependent exocytosis. Moreover, transfection with wild-type RalA, but not RalA(E38R), enhanced GTP-dependent exocytosis. In contrast the RalA and the Sec5 fragment showed no inhibition of Ca2+-dependent exocytosis, but cleavage of a SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein by Botulinum neurotoxin blocked both GTP- and Ca2+-dependent exocytosis. Our results indicate that the interaction between RalA and the exocyst complex (containing Sec5) is essential for GTP-dependent exocytosis. Furthermore, GTP- and Ca2+-dependent exocytosis use different sensors and effectors for triggering exocytosis whereas their final fusion steps are both SNARE-dependent.