David K. Apps

Identification and Characterization of a Novel 9.2-kDa Membrane Sector-associated Protein of Vacuolar Proton-ATPase from Chromaffin Granules

Vacuolar proton-translocating ATPase (holoATPase and free membrane sector) was isolated from bovine chromaffin granules by blue native polyacrylamide gel electrophoresis. A 5-fold excess of membrane sector over holoenzyme was determined in isolated chromaffin granule membranes. M9.2, a novel extremely hydrophobic 9.2-kDa protein comprising 80 amino acids, was detected in the membrane sector. It shows sequence and structural similarity to Vma21p, a yeast protein required for assembly of vacuolar ATPase. A second membrane sector-associated protein (M8-9) was identified and characterized by amino-terminal protein sequencing.

Functional and spatial segregation of secretory vesicle pools according to vesicle age

Synaptic terminals and neuroendocrine cells are packed with secretory vesicles, only a few of which are docked at the plasma membrane and readily releasable. The remainder are thought to constitute a large cytoplasmic reserve pool awaiting recruitment into the readily releasable pool (RRP) for exocytosis. How vesicles are prioritized in recruitment is still unknown: the choice could be random, or else the oldest or the newest ones might be favoured. Here we show, using a fluorescent cargo protein that changes colour with time, that vesicles in bovine adrenal chromaffin cells segregate into distinct populations, based on age. Newly assembled vesicles are immobile (morphologically docked) at the plasma membrane shortly after biogenesis, whereas older vesicles are mobile and located deeper in the cell. Different secretagogues selectively release vesicles from the RRP or, surprisingly, selectively from the deeper cytoplasmic pool. Thus, far from being equal, vesicles are segregated functionally and spatially according to age.

Multi-dimensional time-correlated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM) to detect FRET in cells.

We present a novel, multi‐dimensional, time‐correlated single photon counting (TCSPC) technique to perform fluorescence lifetime imaging with a laser‐scanning microscope operated at a pixel dwell‐time in the microsecond range. The unsurpassed temporal accuracy of this approach combined with a high detection efficiency was applied to measure the fluorescent lifetimes of enhanced cyan fluorescent protein (ECFP) in isolation and in tandem with EYFP (enhanced yellow fluorescent protein). This technique enables multi‐exponential decay analysis in a scanning microscope with high intrinsic time resolution, accuracy and counting efficiency, particularly at the low excitation levels required to maintain cell viability and avoid photobleaching. Using a construct encoding the two fluorescent proteins separated by a fixed‐distance amino acid spacer, we were able to measure the fluorescence resonance energy transfer (FRET) efficiency determined by the interchromophore distance. These data revealed that ECFP exhibits complex exponential fluorescence decays under both FRET and non‐FRET conditions, as previously reported. Two approaches to calculate the distance between donor and acceptor from the lifetime delivered values within a 10% error range. To confirm that this method can be used also to quantify intermolecular FRET, we labelled cultured neurones with the styryl dye FM1‐43, quantified the fluorescence lifetime, then quenched its fluorescence using FM4‐64, an efficient energy acceptor for FM1‐43 emission. These experiments confirmed directly for the first time that FRET occurs between these two chromophores, characterized the lifetimes of these probes, determined the interchromophore distance in the plasma membrane and provided high‐resolution two‐dimensional images of lifetime distributions in living neurones.

Cytochrome b561 is identical with chromomembrin B, a major polypeptide of chromaffin granule membranes

Abstract Cytochrome b 561 from bovine chromaffin granules is identical with chromomembrin B, a major membrane protein of hitherto unknown function. The cytochrome was solubilized with a mixture of non-ionic and anionic detergents, and purified by ammonium sulphate fractionation and hydrophobic column chromatography: it was shown to have an apparent molecular weight on dodecylsulphate gel electrophoresis of 22,000, and an isoelectric point of 6.2. Haem: protein ratios in different preparations of the cytochrome were 26–40 nmol/mg protein. Two-dimensional electrophoretic ‘maps’ of the purified cytochrome, of granule membranes and of granule matrix proteins are presented. Since chromomembrin B is known to extend across the chromaffin granule membrane, cytochrome b 561 could be involved in transmembrane electron transfer.

Isolation and characterization of magnesium adenosinetriphosphatase from the chromaffin granule membrane

1. Introduction Catecholamine storage vesicles (chromaffin granules) of the bovine adrenal medulla have long been thought to contain a Mg-ATPase (EC 3.6.1.3 .) [ 1,2] . Although the association of this activity with chro- maffin granules has recently been questioned [3] , its co-purification with the granule membrane has been reported [4] . There is considerable evidence that ATP hydrolysis by chromaffin granules and resealed ‘ghosts’ results in acidification of the interior of the vesicle [5-71 and the uptake of catecholamines in response to transmembrane pH-gradients has been demonstrated in resealed chromaffin granule ‘ghosts’ (Phillips, J. H., personal communication). In the present study we report the isolation of a soluble form of Mg-ATPase from chromaffin granule mem- branes and an investigation of its structure and properties. Electrophoretic analysis suggests the presence of at least 3 types of subunit, which have similar electrophoretic mobility to the major subunits of mitochondrial ATPase. However, the enzymes from mitochondria and chromaffin granules differ in their sensitivity to some inhibitors.

Biosynthetic relationship between the major matrix proteins of adrenal chromaffin granules

The matrix of the chromaffin granule contains a family of acidic proteins, collectively known as the chromogranins. It has been suggested that this family results protease action on the major component, chromogranin A. Evidence for this has now been obtained from in vitro translation of adrenal medullary messenger RNA and immunoprecipitation of translation products using an antiserum directed against chromogranin A, but which also recognises other chromogranins.

Both the transmembrane pH gradient and the membrane potential are important in the accumulation of amines by resealed chromaffin‐granule ‘ghosts’

The secretory granules of the adrenal medulla, known as chromaffin granules, contain high concentrations of catecholamines and ATP, associated with soluble proteins (reviewed [ 11). Catecholamines are transported into the granule matrix by a reserpinesensitive permease, which shows specificity for the (-) forms of adrenaline and noradrenaline, and also transports dopamine and S-hydroxytryptamine [2]. Accumulation of these substrates by intact granules is driven by ATP, and it is well established [3-S] that an ATPase in the chromaffin granule membrane translocates protons into the granule; this results in creation of a transmembrane proton gradient (ApH) and potential (A

Unusual redox behaviour of cytochrome b-561 from bovine chromaffin granule membranes

), amine uptake being dependent upon these, rather than on ATP hydrolysis itself. Details of the mechanism by which amine uptake is coupled to the collapse of ApH and/or A

Isolation of a DCCD-binding protein from bovine chromaffin-granule membranes.

have not, however, been elucidated; we now report some studies on the initial rates of uptake of noradrenaline, dopamine and 5-hydroxytryptamine which suggest that transport can be driven by an imposed ApH, in the absence of A

Rapid purification and reconstitution of a plant vacuolar ATPase using triton X-114 fractionation : subunit composition and substrate kinetics of the H+-ATPase from the tonoplast of Kalanchoë daigremontiana

, but that the rate of uptake is increased by superimposition of a membrane potential.