James G. Pryde

Caspase-mediated cleavage of the stacking protein GRASP65 is required for Golgi fragmentation during apoptosis

The mammalian Golgi complex is comprised of a ribbon of stacked cisternal membranes often located in the pericentriolar region of the cell. Here, we report that during apoptosis the Golgi ribbon is fragmented into dispersed clusters of tubulo-vesicular membranes. We have found that fragmentation is caspase dependent and identified GRASP65 (Golgi reassembly and stacking protein of 65 kD) as a novel caspase substrate. GRASP65 is cleaved specifically by caspase-3 at conserved sites in its membrane distal COOH terminus at an early stage of the execution phase. Expression of a caspase-resistant form of GRASP65 partially preserved cisternal stacking and inhibited breakdown of the Golgi ribbon in apoptotic cells. Our results suggest that GRASP65 is an important structural component required for maintenance of Golgi apparatus integrity.

Triton X-114: a detergent that has come in from the cold

Abstract The integral proteins of membranes rich in cholesterol and phospholipid can be fractionated by a temperature-induced phase separation in Triton X-114 into three classes differing in their apparent hydrophobicity and free from contamination by adsorbed soluble protein.

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.

Temperature-dependent Arrest of Neutrophil Apoptosis FAILURE OF Bax INSERTION INTO MITOCHONDRIA AT 15 °C PREVENTS THE RELEASE OF CYTOCHROME c

Apoptosis is essential for the resolution of neutrophilic inflammation. To define the mechanisms triggering the execution phase of apoptosis we developed and utilized a model in which culture of human neutrophils at 15 °C for 20 h arrested apoptosis and subsequent warming to 37 °C triggered a synchronous burst of apoptosis. Treatment of 15 °C cultured neutrophils with the pan-caspase inhibitor zVAD-fmk just before warming to 37 °C inhibited the morphological changes associated with apoptosis, but did not prevent the insertion of the proapoptotic protein Bax into mitochondria nor the inhibition of secretion and the externalization of phosphatidylserine, indices of neutrophil apoptosis. In both intact neutrophils and a cell-free extract, cytochrome c released from mitochondria induced proteolytic cleavage of procaspase-3. At 15 °C the binding of Bax to mitochondria was uncoupled from Bax insertion into the mitochondrial membrane required for the release of cytochrome c. Apoptosis was also inhibited by low pH during warming to 37 °C, suggesting that changes to the conformation of Bax, necessary for membrane insertion, were being inhibited. Bax insertion was only sensitive to zVAD-fmk when added at the start of the 15 °C culture period, suggesting that a cytoplasmic substrate of the effector caspases may mediate in the mechanism of Bax insertion into mitochondria.

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

Constitutive neutrophil apoptosis in culture is modulated by cell density independently of β2 integrin-mediated adhesion

), 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

Glycoproteins of the Chromaffin Granule Membrane: Separation by Two-Dimensional Electrophoresis and Identification by Lectin Binding

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

OKADAIC ACID INDUCES SELECTIVE ARREST OF PROTEIN TRANSPORT IN THE ROUGH ENDOPLASMIC RETICULUM AND PREVENTS EXPORT INTO COPII-COATED STRUCTURES

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

The Chromaffin Granule: A Model System for the Study of Hormones and Neurotransmittersa

Although inflammatory mediators modulate the rate of constitutive neutrophil apoptosis in vitro the effects of micro‐environmental conditions have not been fully investigated. In this study, we demonstrate that the rate of constitutive neutrophil apoptosis is affected by the number of cells per unit surface area, with enhanced survival at high cell density. Furthermore, the presence of protein or serum in the culture medium also enhances neutrophil survival. These effects were independent of β2 integrin‐mediated adhesion and were not influenced by specific adhesion to extracellular matrix components. Thus, the rate of neutrophil apoptosis is fundamentally influenced by micro‐environmental conditions and indicates that factors such as cell density and extracellular protein concentration must be considered when investigating mechanisms regulating inflammatory cell apoptosis in vitro.

NF-κB Activation Is a Critical Regulator of Human Granulocyte Apoptosis in Vitro

Abstract: The proteins of highly purified chromaffin granule membranes were separated by one or two‐dimensional electrophoresis, then transferred to nitrocellulose sheets; glycosylation was investigated by binding of several different radioiodinated lectins. Over 20 different glycosylated components were identified; comparison with mitochondrial and microsomal fractions suggested that most of the major glycoproteins are genuine components of the chromaffin granule membrane, rather than contaminants originating in other organdies. Two dimensional electrophoresis revealed heterogeneity within several of the glycoproteins, and this is ascribed to differences in the state of glycosylation, on the basis of shifts in electrophoretic mobility produced by treatment with neuraminidase. Neuraminidase treatment of chromaffin granule membranes also enhances the binding of many lectins. The identities of the lectin‐binding bands are discussed: neither cytochrome b561 nor the F1‐like ATPase appears to be glycosylated. Chromogranin A, although a glycoprotein, does not bind any of the lectins tested, but a number of concanavalin‐A binding proteins, as well as dopamine β‐hydroxylase, are present in the chromaffin granule lysate.