Aggregation Of Chromaffin Granules Research Articles

Lipid Segregation and Membrane Budding Induced by the Peripheral Membrane Binding Protein Annexin A2*

The formation of dynamic membrane microdomains is an important phenomenon in many signal transduction and membrane trafficking events. It is driven by intrinsic properties of membrane lipids and integral as well as membrane-associated proteins. Here we analyzed the ability of one peripherally associated membrane protein, annexin A2 (AnxA2), to induce the formation of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-rich domains in giant unilamellar vesicles (GUVs) of complex lipid composition. AnxA2 is a cytosolic protein that can bind PI(4,5)P2 and other acidic phospholipids in a Ca(2+)-dependent manner and that has been implicated in cellular membrane dynamics in endocytosis and exocytosis. We show that AnxA2 binding to GUVs induces lipid phase separation and the recruitment of PI(4,5)P2, cholesterol and glycosphingolipids into larger clusters. This property is observed for the full-length monomeric protein, a mutant derivative comprising the C-terminal protein core domain and for AnxA2 residing in a heterotetrameric complex with its intracellular binding partner S100A10. All AnxA2 derivatives inducing PI(4,5)P2 clustering are also capable of forming interconnections between PI(4,5)P2-rich microdomains of adjacent GUVs. Furthermore, they can induce membrane indentations rich in PI(4,5)P2 and inward budding of these membrane domains into the lumen of GUVs. This inward vesiculation is specific for AnxA2 and not shared with other PI(4,5)P2-binding proteins such as the pleckstrin homology (PH) domain of phospholipase Cδ1. Together our results indicate that annexins such as AnxA2 can efficiently induce membrane deformations after lipid segregation, a mechanism possibly underlying annexin functions in membrane trafficking.

Transcription, biochemistry and localization of nematode annexins.

The transcription of three annexin genes in the nematode, Caenorhabditis elegans, was detected by reverse transcriptase/polymerase chain reaction amplification of messenger RNAs. The highest level of expression was from the nex-1 gene, with lower levels detected for the nex-2 and nex-3 genes. The expression of nex-1 was reduced in the Dauer larval stage relative to the other annexins, correlating with the absence of the spermathecal valves, a major site of nex-1 protein localization. Recombinant nex-1 protein was expressed in yeast, isolated by calcium-dependent binding to acidic phospholipids, and its membrane binding and aggregating activities characterized using bovine chromaffin granules as a representative intracellular substrate. Binding to granule membranes was promoted by calcium with half-maximal binding seen at 630 microM calcium. Chromaffin granule aggregation was similarly promoted by the nex-1 protein at 630 microM calcium. This low sensitivity to calcium suggests the annexin can only be activated in vivo near the plasma membrane or other sources of calcium. Sequences including the nex-1 promoter were fused to the gene for green fluorescent protein and this construct was introduced into nematodes by microinjection. Examination of transgenic offspring revealed specific nex-1 promoter activity in the pharynx, the hypodermal cells, the vulva, and the spermathecal valve, locations in which the annexin may function in collagen secretion/deposition and membrane-membrane interactions. A sensitive anti-nex-1 antibody labelled with rhodamine was injected into body cavities of the nematode but did not detect extracellular nex-1 protein. Therefore, this annexin is apparently cytosolic and may function on the cytoplasmic side of the plasma membrane of the spermathecal valve to chaperon the folding of this membrane during the opening and closing of the valve.

Highly Sensitive and Stable Phosphatidylserine Liposome Aggregation Assay for Annexins

Annexins are a gene family of Ca2+-dependent membrane binding proteins which interact specifically with acidic phospholipids. We describe here details of highly sensitive and precise assays for annexins I and V, utilizing turbidometric analysis of phosphatidylserine liposome aggregation. In the case of annexin I, the new assay is 7-fold more sensitive than the conventional chromaffin granule aggregation assay in terms of threshold for detection and the rate of increase of initial absorbance is 15-fold greater. Annexin V, which binds but does not aggregate liposomes, can be assayed on the basis of inhibition of Ca2+-dependent liposome aggregation. Further comparative advantages of the assay include lower expense and increased shelf life of the liposome reagent.

Calcium-dependent Binding of Sorcin to the N-terminal Domain of Synexin (Annexin VII)

The annexins are characterized by their ability to bind phospholipid membranes in a Ca2+-dependent manner. Sequence variability between the N-terminal domains of the family members may contribute to the specific cellular function of each annexin. To identify proteins that interact with the N-terminal domain of synexin (annexin VII), a fusion protein was constructed composed of glutathione S-transferase fused to amino acids 1-145 of human synexin. Affinity chromatography using this construct identified sorcin as a Ca2+-dependent synexin-binding protein. Overlay assays confirmed the interaction. The glutathione S-transferase construct associates with recombinant sorcin over the range of pCa2+ = 4.7-3.1 with no binding observed at pCa2+ = 5.4. Overlay assays using deletion constructs of the synexin N-terminal domain mapped the sorcin binding site to the N-terminal 31 amino acids of the synexin protein. Additionally, synexin forms a complex with sorcin and recruits this protein to chromaffin granule membranes in a Ca2+-dependent manner. Sorcin is able to inhibit synexin-mediated chromaffin granule aggregation in a manner saturable with increasing sorcin concentrations, but does not influence the Ca2+ sensitivity of synexin-mediated granule aggregation. Therefore, the interaction between sorcin and synexin may serve to regulate the functions of these proteins on membrane surfaces in a Ca2+-dependent manner.

Characterization of human recombinant annexin II tetramer purified from bacteria: role of N-terminal acetylation.

Annexin II tetramer (AIIt) is a Ca2+-dependent, phosphatidylserine-binding, and F-actin-bundling phosphoprotein which is localized to both the extracellular and cytoplasmic surfaces of the plasma membrane. The tetramer is composed of two p36 heavy chains and two p11 light chains. We have produced prokaryotic cDNA expression constructs for both p36 and p11. Both proteins were expressed in large amounts in Escherichia coli upon induction with IPTG. Electrospray ionization mass spectrometry and amino acid sequence analysis of purified recombinant p36 (rp36) and recombinant p11 (rp11) suggested that the recombinant proteins were identical to their native counterparts except for the lack of N-terminal acetylation of rp36. Furthermore, the non-acetylated rp36 bound rp11 and formed AIIt. The circular dichroism spectra and urea denaturation profiles of acetylated AIIt and non-acetylated rAIIt were identical. In addition, both the acetylated AIIt and non-acetylated rAIIt were similar in their Ca2+ dependence and concentration dependence of phospholipid liposome aggregation, chromaffin granule aggregation, and F-actin bundling. These results suggest that N-terminal acetylation of p36 is not in fact necessary for binding of the protein to p11 and that N-terminal acetylation does not affect the conformational stability of AIIt or the in vitro activities of AIIt. The availability of large amounts of rAIIt will facilitate further characterization of the structure-function relationships of the protein.

"In Vitro" Phosphorylation of Annexin 2 Heterotetramer by Protein Kinase C: COMPARATIVE PROPERTIES OF THE UNPHOSPHORYLATED AND PHOSPHORYLATED ANNEXIN 2 ON THE AGGREGATION AND FUSION OF CHROMAFFIN GRANULE MEMBRANES

Heterotetrameric annexin 2 phosphorylated "in vitro" by rat brain protein kinase C is purified and obtained devoid of unphosphorylated protein; it contains 2 mol of phosphate/mol of heterotetramer. The aggregative and binding properties of the phosphorylated annexin 2 toward purified chromaffin granules are compared with those of the unphosphorylated annexin 2. Annexin 2 binds to chromaffin granules with high affinity. Phosphorylation of annexin 2 decreases the affinity of this binding without affecting the maximum binding capacity. The binding curves are strongly cooperative. It is suggested that a surface oligomerization of the proteins may take place upon binding. Besides, phosphorylation of annexin 2 is followed by a dissociation of the light chains from the heavy chains in the heterotetramer. Whereas annexin 2 induces the aggregation of chromaffin granules at microM calcium concentration, the phosphorylated annexin 2 does not induce aggregation at any concentration of calcium either at pH 6 or 7. The phosphorylation of annexin 2 by protein kinase C, MgATP, and 12-O-tetradecanoylphorbol-13-acetate on chromaffin granules induces a fusion of chromaffin granules membranes observed in electron microscopy. The fusion requires the activation of protein kinase C by 12-O-tetradecanoylphorbol-13-acetate. Given these results and since annexin 2 is phosphorylated by protein kinase C under stimulation of chromaffin cells, it is suggested that phosphorylated annexin 2 may be implicated in the fusion step during exocytosis of chromaffin granules.

Salt dependency of chromaffin granule aggregation by annexin II tetramer.

Annexin II tetramer (AIIt) is a Ca2+ and phospholipid binding protein that has been shown to reconstitute secretion in permeabilized adrenal medulla cells. In the present study, we have characterized the interactions of AIIt with biological membranes using isolated adrenal medulla secretory granules as a model system. Without added salt, maximal binding of AIIt to chromaffin granules occurred in the absence of AIIt-dependent chromaffin granule aggregation, whereas increasing the osmolality of the reaction mixture with sucrose did not activate AIIt-dependent chromaffin granule aggregation. As the KCl or potassium glutamate concentration of the reaction mixture was increased to between 30 and 50 mM salt, AIIt-dependent chromaffin granule aggregation increased to a maximum, while AIIt binding to chromaffin granules decreased. As the salt concentration was increased from 50 to 150 mM, both AIIt-dependent chromaffin granule aggregation and the binding of AIIt to chromaffin granules were decreased. Furthermore, at optimal salt concentration, KCl and potassium glutamate activated AIIt-dependent aggregation of chromaffin granules to maximum values of about 210% and 195% of control, respectively, whereas potassium phosphate supported AIIt-dependent aggregation of chromaffin granules to only 120% of control. The concentration of AIIt for half-maximal binding to chromaffin granules without added salt or at 50 mM KCl was 0.163 +/- 0.007 (mean +/- SD, n = 3) or 0.173 +/- 0.034 microM AIIt (mean +/- SD, n = 3), respectively, and binding of AIIt to chromaffin granules was not measurable at 150 mM KCl. In contrast, at 50 mM KCl, half-maximal AIIt-dependent chromaffin granule aggregation required 0.171 +/- 0.001 microM AIIt (mean +/- SD, n = 3) and was not measurable without added salt or in the presence of 150 mM KCl. Without added salt, at 50 mM KCl, or at 150 mM KCl, the Ca2+ concentrations for half-maximal aggregation of chromaffin granules and the maximal extent of chromaffin granule aggregation (Amax) were pCa2+ = 3.79 +/- 0.062 (mean +/- SD, n = 3) and Amax = 127% of control, pCa2+ = 6.07 +/- 0.021 (mean +/- SD, n = 3) and Amax = 185% of control, or pCa2+ 4.41 +/- 0.07 (mean +/- SD, n = 3) and Amax = 156% of control, respectively. The stimulation of chromaffin granule aggregation activity and the chromaffin granule binding activity of AIIt was reversible by removal of Ca2+. These results suggest that both ionic strength and salt composition modulate both AIIt-dependent chromaffin granule aggregation and binding to the membranes of these secretory granules.

Role of the amino-terminal domain in regulating interactions of annexin I with membranes: Effects of amino-terminal truncation and mutagenesis of the phosphorylation sites

Phosphorylation of the N-terminal tail by protein kinase C strongly inhibits the ability of bovine or human annexin I to aggregate chromaffin granules by increasing the calcium requirement 4-fold (Wang, W., & Creutz, C. E. (1992) Biochemistry 31, 9934-9936). In the present study three forms of human annexin I truncated in the amino terminus at residue Trp-12, Lys-26, or Lys-29 exhibit dramatic differences in their sensitivities to calcium in a chromaffin granule aggregation assay, while the [Ca2+](1/2)max values for binding of the truncated proteins to granule membranes are similar. Cleavage at Trp-12 causes a 3-fold decrease in calcium sensitivity in the membrane aggregation assay, while cleavage at Lys-26 causes a 4-fold enhancement of calcium sensitivity. In contrast, cleavage at Lys-29 results in virtually no change in calcium sensitivity. Mutagenic substitution with negatively charged amino acids of Ser-27, a site for phosphorylation by protein kinase C, or Tyr-21, a site for phosphorylation by the epidermal growth factor receptor kinase, mimics the inhibition of granule-aggregating activity seen with phosphorylation by protein kinase C. When bovine chromaffin cells are stimulated to secrete by nicotine, annexin I is phosphorylated in the amino terminus. Thr-24 and Ser-28, which are sites for phosphorylation by protein kinase C in vitro, are two of the sites phosphorylated in vivo in stimulated chromaffin cells. These data demonstrate that the ability of annexin I to promote membrane aggregation is highly sensitive to changes in the structure of the N-terminal domain of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential recognition of secretory vesicles by annexins

The interactions of synexin, calpactin, and p68 (annexins VII, II, and VI) with zymogen granules and adrenal medullary chromaffin granules were compared. Synexin promoted the aggregation and fusion of both types of organelles. Calpactin inhibited the action of synexin on zymogen granules but would aggregate only chromaffin granules. p68 inhibited aggregation of chromaffin granules by calpactin and fusion of zymogen granules by synexin. The results demonstrate that these annexins, all of which bind acidic phospholipids, interact differently with different biological membranes and therefore may play specific and non-overlapping roles in cells where they are coexpressed.

The participation of annexin II (calpactin I) in calcium-evoked exocytosis requires protein kinase C.

Permeabilized adrenal chromaffin cells secrete catecholamines by exocytosis in response to micromolar calcium concentrations. Recently, we have demonstrated that chromaffin cells permeabilized with digitonin progressively lose their capacity to secrete due to the release of certain cytosolic proteins essential for exocytosis (Sarafian T., D. Aunis, and M. F. Bader. 1987. J. Biol. Chem. 34:16671-16676). Here we show that one of the released proteins is calpactin I, a calcium-dependent phospholipid-binding protein known to promote in vitro aggregation of chromaffin granules at physiological micromolar calcium levels. The addition of calpactin I into digitonin- or streptolysin-O-permeabilized chromaffin cells with reduced secretory capacity as a result of the leakage of cytosolic proteins partially restores the calcium-dependent secretory activity. This effect is specific of calpactin I since other annexins (p32, p37, p67) do not stimulate secretion at similar or higher concentrations. Calpactin I requires the presence of Mg-ATP, suggesting that a phosphorylating step may regulate the activity of calpactin. Calpactin is unable to restore the secretory activity in cells which have completely lost their cytosolic protein kinase C or in cells having their protein kinase C inhibited by sphingosine or downregulated by long-term incubation with TPA. In contrast, calpactin I prephosphorylated in vitro by purified protein kinase C is able to reconstitute secretion in cells depleted of their protein kinase C activity. This stimulatory effect is also observed with thiophosphorylated calpactin I which is resistant to cellular phosphatases or with phosphorylated calpactin I introduced into cells in the presence of microcystin, a phosphatase inhibitor. These results suggest that calpactin I is involved in the exocytotic machinery by a mechanism which requires phosphorylation by protein kinase C.

Aggregation of chromaffin granules by calpactin at micromolar levels of calcium.

Several cytosolic proteins bind to secretory granule membranes in a Ca2+-dependent manner and thus may be involved in the mediation of membrane interactions during exocytosis. One of these proteins, calpactin, is a tetramer consisting of two heavy chains of relative molecular mass (Mr) 36K (p36) and two light chains of 10K (p10). We report here that calpactin promotes the Ca2+-dependent aggregation and fatty acid-dependent fusion of chromaffin granule membranes at a level of Ca2+ that is lower than that reported for other granule-aggregating proteins, and which parallels the Ca2+ requirement for secretion from permeabilized chromaffin cells. We found subunits of calpactin to be inactive in promoting granule aggregation. Two distinct 33K proteolytic fragments of p36, differing at their N termini, also promote granule aggregation but with different Ca2+ sensitivities from calpactin. These differences suggest that the N-terminal portion of p36 modulates the Ca2+/lipid binding sites in the core portion of p36 (ref.5).

Synhibin: A new calcium-dependent membrane-binding protein that inhibits synexin-induced chromaffin granule aggregation and fusion

We report the isolation and purification of synhibin, a new M r 68000 protein, which inhibits synexin. Synexin mediates Ca 2+-dependent chromaffin granule aggregation and fusion, processes perhaps important during exocytosis. Our data indicate that synhibin action involves competition with synexin for a site on the chromaffin granule membrane involved in membrane contact. Synhibin may thus be an important intracellular regulator of synexin action during secretion.

Specificity of synexin-induced chromaffin granule aggregation

Preparations of synexin (1) exhibit a self-interaction in the absence of chromaffin granules as evidenced by an increase in absorbance at the wave-length used for observing granule aggregation (2). We incorporated this observation into a new formula for calculating the synexin-induced chromaffin granule aggregation. According to this amended analysis, synexin-induced aggregation is specific for chromaffin granules or their membranes. Treatment of intact chromaffin granuleswith trypsin or pronase renders the granules unresponsive to synexin.

Self-association of synexin in the presence of calcium. Correlation with synexin-induced membrane fusion and examination of the structure of synexin aggregates.

It has been proposed (Creutz, C. E., Pazoles, C. J., and Pollard, H. B. (1978) J. Biol. Chem. 253, 2858-2866) that synexin, an adrenal medullary protein that causes Ca2+-dependent aggregation of isolated chromaffin granules, might be the intracellular receptor for Ca2+ in the process of exocytosis. We now report that Ca2+ interacts directly with isolated synexin, inducing rapid self-association. Mg2+ or Sr2+ cannot substitute for Ca2+, and Ba2+ is only weakly effective at stimulating self-association. We have analyzed 90 degree light scattering data to determine a titration curve for the activation of synexin by Ca2+ in the self-association reaction. The curve has a Hill coefficient of 2.3 and is half-maximal at 200 micron Ca2+, which correlates exactly with the Ca2+ titration curve for the aggregation of chromaffin granules by synexin. Electron microscopy of negatively stained samples reveals that synexin monomers associate to form 50 A by 150 A rods which in turn associate side to side and end to end to form bundles of parallel rods. We suggest that synexin exists as a soluble monomer in the cytoplasm of the resting chromaffin cell and that when the cell is stimulated to secrete, the increase in the intracellular concentrations of Ca2+ causes synexin to form rods, similar to those formed in vitro, which bind to chromaffin granule and plasma membranes, joining them together to form the "pentalaminar" fusion complexes characteristic of the first step in exocytosis.

Calcium-induced displacement of membrane-associated particles upon aggregation of chromaffin granules.

Isolated chromaffin granules incubated in 10 millimolar calcium chloride aggregated, forming contact sites with a pentalaminar membrane structure. These circular attachment sites were free of membrane-associated particles, which accumulated at the periphery. Incubation in 20 millimolar ethylenediaminetetraacetic acid reversed these changes, which are regarded as initial events in the membrane fusion reaction.