Thomas C. Südhof

Calelectrin, a Calcium-Dependent Membrane-Binding Protein Associated with Secretory Granules in Torpedo Cholinergic Electromotor Nerve Endings and Rat Adrenal Medulla

Abstract: Calelectrin, a calcium‐dependent membrane‐binding protein of subunit molecular weight 32,000 has been isolated from the electric organ of Torpedo, and shown to occur in cholinergic neurones and in bovine adrenal medulla. In this study a monospecific antiserum against the Torpedo protein has been used to study the localization of calelectrin in the rat adrenal gland. The cortex was not stained, whereas in the medulla the cytoplasm of the chromaffin cells was stained in a particulate manner. An identical staining pattern was obtained with an antiserum against the chromaffin granule enzyme dopamine β‐hydroxylase, although the two antisera did not cross‐react with the same antigen. The purified protein aggregates bovine chromaffin granule membranes and cholinergic synaptic vescicles and also self aggregates in a calcium‐dependent manner. Negative staining results demonstrate that calcium induces a transformation of the purified protein from circular structures 30–80 nm in diameter into a highly aggregated structure. Calelectrin may have a structural or regulatory role in the intracellular organization of secretory cells.

Characterization of Calelectrin, a Ca2+-Binding Protein Isolated from the Electric Organ of Torpedo marmorata

Abstract: We report a fast (< 1 day) and efficient (2–3 mg protein/100 g tissue) isolation method for calelectrin, a protein of Mr 34,000 in the electric organ of Torpedo marmorata that binds to membranes in the presence of Ca2+. Purified protein was used to investigate the nature of its interaction with membranes and with Ca2+. Calelectrin binds to liposomes composed of total extractable lipids from the electric organ in a Ca2+‐dependent and ‐specific manner with half‐maximal binding between 3 and 7 μM free Ca2+. This binding is totally inhibited by 1 mM mercaptoethanol. It is also shown that calelectrin directly binds Ca2+ in solution by two techniques: at 1 and 10 μM Ca2+ it binds 45Ca2+ as measured by gel permeation chromatography, and it contains saturable Tb3+‐binding sites that are Ca2+‐displaceable. An investigation of the proteins endogenous fluorescence shows that although it contains both tryptophan and tyrosine, there is no change in the apparent quantum yield as a function of Ca2+. Ca2+‐dependent hydrophobic affinity chromatography of the total soluble proteins from Torpedo electric organ shows that Torpedo calelectrin, like calmodulin and mammalian calelectrins, is specifically retained in the presence of Ca2+ and eluted by EGTA. Calelectrin also contains high‐affinity sites for hydrophobic fluorescence probes such as N‐phenyl‐1‐naphthylamine, 2‐CP‐toluidinylnaphthalene‐6‐sulfonic acid, and 1‐anilinonaphthalene‐8‐sulfonic acid, which again unlike calmodulin, show no changes as a function of Ca2+. We conclude that calelectrin is a Ca2+‐binding protein whose binding to the lipid moieties of membranes is regulated by physiological changes in the Ca2+ concentration. This binding must be due to specific mechanisms other than simple exposure of hydrophobic sites.

Core structure, internal osmotic pressure and irreversible structural changes of chromaffin granules during osmometer behaviour

In the adrenal medullary cells, catecholamines are stored in and secreted from specialized secretory vesicles, the chromaffin granules. In order to gain some understanding of both functions of chromaffin granules, it is important to characterize their biophysical organization. Using isolated bovine chromaffin granules we have investigated the osmometer behaviour of chromaffin granules by 31P-NMR and fluorescence spectroscopy, by turbidity measurements and by electron-microscopic determination of chromaffin granule size distributions. On the basis of the osmometer model we have formulated equations predicting the behaviour of the native catecholamine fluorescence quenching and of the size of chromaffin granules a a function of osmolarity and have shown experimentally that the granules behaviour conforms to these. It was possible to estimate the osmotic activity of the chromaffin granule core solution and the mean absolute water space in chromaffin granules from the determination of the size distributions as a function of osmotic pressure. With NMR spectroscopy a selective line-broadening of the alpha-resonances was observed with increasing osmolarities, while the gamma-phosphorus resonances remained virtually unchanged. Possibly there is an increase in core viscosity with osmolarity which affects only the alpha- and beta-phosphorus groups. While suspending chromaffin granules from lower to higher osmolarities causes no lysis, moving them back to their original osmolarity at which they were previously stable lyses them, thereby releasing a maximum of 70% of their releasable protein. This hyperosmolar lysis is independent of preincubation times in the higher osmolarities and of the absolute dilution applied but depends on dilution beyond the 405 to 322 mosM sucrose range. Under the experiment conditions no uptake of sucrose from the medium into the granules could be measured, thereby suggesting that hyperosmolar lysis is a phenomenon not due to solute penetration. Since with NMR and fluorescence spectroscopy no chemical changes in the core composition can be observed, we conclude that hyperosmolar lysis may be caused by irreversible membrane relaxation upon osmotic shrinking.

Calelectrins are a ubiquitous family of Ca2+-binding proteins purified by Ca2+-dependent hydrophobic affinity chromatography by a mechanism distinct from that of calmodulin

The calelectrins, a heterogeneous group of three new Ca2+-binding proteins of M 67 000, 35 000 and 32 500, copurify with calmodulin during Ca2+-dependent hydrophobic affinity chromatography (Südhof et al., Biochemistry, in press, 1984). This property is exploited for the rapid purification of all three calelectrins including for the first time the Mr 35 000, from commercially available acetone powders from several bovine tissues (heart, liver, brain, pancreas and testis). The nature of the Ca2+-dependent interaction of the calelectrins with hydrophobic affinity matrices has been investigated. As with calmodulin, the Ca2+-binding sites of all three purified calelectrins can be probed with Tb3+ which binds to them in a stoichiometric, saturable and Ca2+-displaceable manner. However, using several hydrophobic fluorescence probes which bind to the proteins, contrary to calmodulin no Ca2+-dependent exposure of hydrophobic sites could be detected in any of the three purified proteins. Therefore the Ca2+-dependent purification of the calelectrins on hydrophobic affinity columns seems not to involve the surface exposure of hydrophobic sites and the calelectrins have in this respect little similarity to calmodulin.

Calcium-promoted resonance energy transfer between fluorescently labeled proteins during aggregation of chromaffin granule membranes

Proteins of the chromaffin granule membrane were covalently labeled in situ with sulfhydryl-specific fluorophores. Using MIANS (maleimide iodoaminonaphthyl sulfonate) as the donor and fluorescein mercury acetate or fluorescein-5-maleimide as the acceptor. Förster fluorescence resonance energy transfer (FRET) could be employed to measure the degree of inter-membrane and intra-membrane protein-protein contact upon Ca2+-induced aggregation of the membranes. The four major findings were: (1) Raising the Ca2+ concentration to approx. 500 microM causes the proteins to aggregate in the plane of the membrane. This is demonstrated by Ca2+-induced increases in the fluorescence resonance energy transfer in double labeled membranes. This effect is not protein-concentration dependent and occurs at calcium concentrations too low for granule aggregation, implying intra-membrane protein clustering or patching. To our knowledge this is the first direct demonstration of the fluid mosaic nature of subcellular organelles. (2) If two sets of granules are labeled separately, Ca2+-induced aggregation brings at least 74% of the labeled proteins into close transmembrane proximity. This effect is also observed at 10-100-fold slower rates in the absence of calcium and can be greatly reduced by depleting the granule membrane of labeled peripheral proteins. It is enhanced if the granules are aggregated by Ca2+ or K+. We conclude that (some) peripheral proteins can transfer from one membrane surface to another. (3) Aggregation of separately labeled sets of membranes by Ca2+ also produces transmembrane energy transfer since: (a) the Km for Ca2+-induced quantum transfer is in the same range as the Km for aggregation; (b) the reaction is protein-concentration dependent; (c) reversal of aggregation also (partially) reverses donor quenching. (4) A kinetic analysis of the transmembrane effect shows it to be 5-10-fold slower than aggregation itself, supporting earlier suggestions (Haynes, D.H., Kolber, M. and Morris, S.J., (1979) J. Theor. Biol. 81, 713-743) that lipid and protein rearrangements are secondary to granule membrane aggregation.

The chromaffin granule as a model for membrane fusion: implications for exocytosis

Rapid freeze/freeze-fracture and thin section electron micrographic studies of the Ca2+-promoted aggregation and fusion of isolated bovine adrenal medullary chromaffin granule membranes show that the granules undergo a series of morphological changes. The contact region becomes quite extensive and the membrane curvature changes radically at the edge of the contact site. The core material retracts away from the contact site leaving an electron lucent stripe; however, it remains adjacent to the membrane in the non-contact areas. The pentalaminar double membrane of the contact region often shows breaks. Close examination reveals that the two granule membranes have fused and become one continuous membrane. Rearrangement of large membrane associated particles (MAPs) can be seen by freeze fracture after Ca2+-promoted granule-granule contact. The broken pentalaminar septum becomes smaller and may break down into globular structures. These observations suggest a series of reactions in which the granules first form an encounter complex, then a stable complex. The membranes within the contact region undergo lateral displacement of the proteins and phase separations of the lipids, and then fuse. Analysis of the kinetics of turbidity and fluorescence changes during granule aggregation and fusion support the main postulates of the model. The initial events of aggregation are facilitated by putative recognition proteins and K+ will promote all activities except fusion. Recent observations that several soluble proteins (synexin, and albumin) will act as fusogens are discussed in terms of the relevance to exocytosis in vivo.

Characteristics and determinants of osmotic lysis in chromaffin granules

(1) Using isolated bovine chromaffin granules, we demonstrate that osmotic lysis is not a random process and establish the osmotic pressure dependence of osmotic lysis in chromaffin granules, the so-called osmotic fragility curve. (2) We show by measuring the release of constituents of the granule core and correlating these with changes in spectroscopic parameters (turbidity and endogenous catecholamine fluorescence), that the latter can be safely used to measure lysis. (3) Within a particular granule population, noradrenaline granules lyse at higher osmolarities than adrenaline granules, suggesting a higher core osmolarity of the noradrenaline granules. (4) The size distribution of chromaffin granules as a function of lysis was determined by the use of whole mount electron microscopy. It is shown that the mean size of chromaffin granules decreases as a function of lysis. (5) On the basis of theoretical considerations three alternative models of the sequence of osmotic lysis in chromaffin granules are proposed. The experimental results best support a model which postulates that during partial osmotic lysis, granule membranes reseal into smaller vesicles after graded release of contents. The osmotic fragility would represent several cycles of lysis and resealing and would not be a reflection of the distribution of osmotic pressures in the granule population.

Evidence for a divalent cation dependent catecholamine storage complex in chromaffin granules

Chromaffin granules, the secretory vesicles of the adrenal medulla, are stable in isotonic sucrose solutions at room temperature; however, when low concentrations of the ionophore A23187 are added, rapid lysis ensues which is dependent on the presence of a divalent cation chelator and is prevented by the addition of either Ca2+ or Mg2+. As little as 10 microM Ca2+ totally inhibit lysis of chromaffin granules by A23187, while 28 mM KCl have no effect. Lysis by A23187 at 4.7 microM is almost 100% in the presence of EDTA in isotonic sucrose in 1 h and can be suppressed by raising the osmotic strength of the medium with half maximal inhibition at 0.57 M sucrose, demonstrating that A23187 causes osmotic lysis of chromaffin granules as a consequence of the withdrawal of divalent cations from the core solution. Our results strongly suggest that divalent cations are involved in the formation of a ionic complex in the core solution which lowers its effective osmotic pressure.

Temperature-induced lysis of chromaffin granules provides evidence against the two-pool hypothesis of catecholamine storage

The temperature-dependent release of core constituents from isolated chromaffin granules in isotonic sucrose has been a controversial and puzzling phenomenon that has been interpreted either as selective catecholamine efflux from different catecholamine pools or as temperature-dependent lysis. We have analysed the kinetics, temperature dependence and physical basis of this process. Our results demonstrate that, upon increasing the ambient temperature, chromaffin granules show a shift in their osmotic fragility to higher osmolarities, which is linearly dependent on temperature and leads to measurable lysis in 0.26 M buffered sucrose at temperatures above 12 degrees C. It is possible to demonstrate both protein and dopamine beta-hydroxylase release when lysis as a function of temperature is measured in 0.26 M buffered sucrose. Real time measurements of the lysis kinetics were recorded on cassettes and analysed by a computer program for exponential decay kinetics. It is shown that the temperature-dependent lysis proceeds in two separate phases, the fast one of which is associated with temperature-dependent shift in the osmotic fragility curve. It has no characteristics of any exponential decay kinetics. The slow phase, when followed over several hours, leads to complete lysis of the granules in a sigmoidal time course at 30 degrees C. We conclude from the absence of exponentiality that there is no basis on which to assume the existence of different catecholamine pools. The fast phase of temperature-dependent lysis can be best explained as a simple temperature-dependent increase of the granule core solutions osmotic pressure, while the slow phase is probably caused by sucrose permeation into the granules. On the basis of these results, we warn against any efflux experiments measuring the temperature-dependent transmitter release from secretory vesicles with highly concentrated core solutions.