Large-scale purification of calf pancreatic zymogen granule membranes.

A protocol for isolating milligram quantities of highly purified zymogen granule membranes from calf pancreas was developed. The method provides a fivefold enriched zymogen granule fraction that is virtually free from major isodense contaminants, such as mitochondria and erythrocytes. Isolated granules are osmotically stable in isosmotic KCl buffers with half-lives between 90 and 120 min. They display specific ion permeabilities that can be demonstrated using ionophore probes to override intrinsic control mechanisms. A Cl- conductance, a Cl-/anion exchanger, and a K+ conductance are found in the zymogen granule membrane, as previously reported for rat pancreatic, rat parotid zymogen granules, and rabbit pepsinogen granules. Lysis of calf pancreatic secretory granules in hypotonic buffers and subsequent isolation of pure zymogen granule membranes yield about 5-10 mg membrane protein from approximately 1000 ml pancreas homogenate. The purified zymogen granule membranes are a putative candidate for the rapid identification and purification of epithelial Cl- channels and regulatory proteins, since they contain fewer proteins than plasma membranes.     

Proteomic analysis of pancreatic zymogen granules: identification of new granule proteins

The composition of zymogen granules from rat pancreas was determined by LC-MS/MS. Enriched intragranular content, peripheral membrane, and integral membrane protein fractions were analyzed after one-dimensional SDS-PAGE and tryptic digestion of gel slices. A total of 371 proteins was identified with high confidence, including 84 previously identified granule proteins. The 287 remaining proteins included 37 GTP-binding proteins and effectors, 8 tetraspan membrane proteins, and 22 channels and transporters. Seven proteins, pantophysin, cyclic nucleotide phosphodiesterase, carboxypeptidase D, ecto-nucleotide phosphodiesterase 3, aminopeptidase N, ral, and the potassium channel TWIK-2, were confirmed by immunofluorescence microscopy or by immunoblotting to be new zymogen granule membrane proteins.     

Protein concentration in the pancreatic zymogen granule

We have estimated the concentration of protein in the zymogen granule, a major storage depot for secretory protein in the pancreatic acinar cell, in four different ways. Each of these approaches yielded roughly similar values. The protein concentration in the granule is approx. 135-270 mg protein/ml granule volume in rat and rabbit, as compared to an average value for the protein concentration of tissue of 135 mg/g tissue for rabbits and 183 mg/g tissue for rats. This is equivalent to an average molarity for the contained proteins of between 4-9 mM based on an estimated average molecular weight for the mixture of 30 000-40 000. An upper limit for the concentration of protein in these granules can reasonably be set at about double the overall concentration of protein in the non-granule portion of the cell.     

Heterogeneity of the zymogen granule membranes in rat pancreas

Zymogen granules (ZG) of rat pancreas have been isolated by the procedure of Paquet et al. The granules lysed when exposed to alkaline pH (pH 8.2), and their membranes could be subfractionated by centrifugation on a sucrose gradient. Four discrete types of membranes corresponding to densities of 1.105, 1.085, 1.075, and 1.020 were obtained, designated types A, B, C, and D, respectively and characterized both by morphological and biochemical criteria. Electrophoretic profiles showed that they contain the same protein bands but in different proportions. Type A membranes are comprised of four major bands corresponding to molecular weights of 80, 69, 54, and 20 kDa, being in higher concentration than the others. Types B and C contain three major bands at 80, 54 and 20 kDa whereas type D is comprised of only two major bands at 69 and 54 kDa, the latter polypeptide corresponding to ATP-diphosphohydrolase activity which is present in all four membrane types. Freeze-fracture of rapidly frozen membranes, followed by transmission electron microscopy (TEM) showed that type A are large superimposed sheets of membranes with amorphous material between sheets. The surface area of these sheets corresponds grossly to the surface of an intact ZG with a few intramembrane particles (IMP) distributed at random or in small aggregates on large smooth fracture planes. Types B and C exhibit a totally different aspect, forming closed vesicles about the size of a small ZG with few IMP distributed at random or in small aggregates on smooth fracture planes. Type D membranes are very small vesicles with no detectable IMP on relatively smooth fracture planes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Organellar proteomics: turning inventories into insights

Subcellular organization is yielding to large-scale analysis. Researchers are now applying robust mass-spectrometry-based proteomics methods to obtain an inventory of biochemically isolated organelles that contain hundreds of proteins. High-resolution methods allow accurate protein identification, and novel algorithms can distinguish genuine from co-purifying components. Organellar proteomes have been analysed by bioinformatic methods and integrated with other large-scale data sets. The dynamics of organelles can also be studied by quantitative proteomics, which offers powerful methods that are complementary to fluorescence-based microscopy. Here, we review the emerging trends in this rapidly expanding area and discuss the role of organellar proteomics in the context of functional genomics and systems biology.     

Modulating zymogen granule formation in pancreatic AR42J cells

Zymogen granules (ZG) are specialized organelles in the exocrine pancreas which allow digestive enzyme storage and regulated secretion. To investigate ZG biogenesis, cargo sorting and packaging, suitable cellular model systems are required. Here, we demonstrate that granule formation in pancreatic AR42J cells, an acinar model system, can be modulated by altering the growth conditions in cell culture. We find that cultivation of AR42J cells in Panserin^? 401, a serum-free medium, enhances the induction of granule formation in the presence or absence of dexamethasone when compared to standard conditions including serum. Biochemical and morphological studies revealed an increase in ZG markers on the mRNA and protein level, as well as in granule size compared to standard conditions. Our data indicate that this effect is related to pronounced differentiation of AR42J cells. To address if enhanced expression of ZG proteins promotes granule formation, we expressed several zymogens and ZG membrane proteins in unstimulated AR42J cells and in constitutively secreting COS-7 cells. Neither single expression nor co-expression was sufficient to initiate granule formation in AR42J cells or the formation of granule-like structures in COS-7 cells as described for neuroendocrine cargo proteins. The importance of our findings for granule formation in exocrine cells is discussed.     

The major protein of pancreatic zymogen granule membranes (GP-2) is anchored via covalent bonds to phosphatidylinositol

GP-2, the major integral protein characteristic of the pancreatic zymogen granule membrane can be released from the membrane by the action of a phosphatidylinositol specific phospholipase C (PI-PLC). In a hydrophobic/hydrophilic phase separation system using the non-ionic detergent Triton X-114, the membrane-bound form of the protein went from the detergent phase into the hydrophilic phase upon action of the phospholipase. PI-PLC solubilization of GP-2 unmasked an antigenic determinant similar to the cross-reacting determinant of the trypanosome variant surface glycoproteins. This determinant being a distinctive feature of the glycan moiety of phosphatidyl-inositol anchored membrane proteins, it established the glycosyl-phosphatidyl-inositol nature of the GP-2 membrane anchor. Since soluble GP-2 is also found in the contents of the granule and is secreted intact into the pancreatic juice, it is likely that one of the mechanisms responsible for its release could be a specific phospholipase. GP-2 is the first glycosyl-phosphatidyl-inositol-anchored protein that is integral to the membrane of an organelle and not located at the surface of the cell.     

Organellar proteomics reveals Golgi arginine dimethylation

The Golgi complex functions to posttranslationally modify newly synthesized proteins and lipids and to sort them to their sites of function. In this study, a stacked Golgi fraction was isolated by classical cell fractionation, and the protein complement (the Golgi proteome) was characterized using multidimensional protein identification technology. Many of the proteins identified are known residents of the Golgi, and 64% of these are predicted transmembrane proteins. Proteins localized to other organelles also were identified, strengthening reports of functional interfacing between the Golgi and the endoplasmic reticulum and cytoskeleton. Importantly, 41 proteins of unknown function were identified. Two were selected for further analysis, and Golgi localization was confirmed. One of these, a putative methyltransferase, was shown to be arginine dimethylated, and upon further proteomic analysis, arginine dimethylation was identified on 18 total proteins in the Golgi proteome. This survey illustrates the utility of proteomics in the discovery of novel organellar functions and resulted in 1) a protein profile of an enriched Golgi fraction; 2) identification of 41 previously uncharacterized proteins, two with confirmed Golgi localization; 3) the identification of arginine dimethylated residues in Golgi proteins; and 4) a confirmation of methyltransferase activity within the Golgi fraction.     

Phosphorylation of zymogen granule membrane proteins in intact rat pancreatic acinar cells

The comparative effects of insulin and ethanolamine on 14CO2 production and lipid synthesis from [U-14C]-D-glucose in isolated rat adipocytes were studied. Ethanolamine (10 mM) increased 14CO2 production (glucose oxidation) about 5-fold and lipogenesis about 3-fold as compared to the control. Ethanolamine was more efficient than 25 microU/ml insulin regarding both parameters, but it was less efficient than 200 microU/ml insulin in glucose oxidation, and equally potent in lipogenesis. The combination of ethanolamine and insulin was more active than insulin alone. The mechanisms of ethanolamine action include facilitation of glucose transport and increase of pyruvate dehydrogenase activity.     

Characterization of phospholipase A2 and acyltransferase activities in purified zymogen granule membranes

Phospholipase A2 and acyltransferase activities were identified in membranes associated with purified pancreatic zymogen granules. In homogenate and granule membranes, phospholipase activity was linearly related to protein concentration and was Ca2(+)-dependent with an alkaline pH optimum. The Ca2+ sensitivity was observed over the range of concentrations through which intracellular ionic Ca2+ is elevated by physiological stimuli in intact cells. Intact zymogen granules and granule membranes also demonstrated reacylating activity in the presence and absence of an exogenous acceptor. Reacylating activity was related to the concentration of lyosphospholipid added and was optimally activated at alkaline pH. A more rapid rate of reacylation was observed when [14C]arachidonoyl CoA was employed as the donor molecule rather than [3H]arachidonate (plus coenzyme A); this suggests the absence of acyl-CoA synthetase in the purified granule membranes. We conclude that granule membrane phospholipase A2 and acyltransferases may be involved in arachidonic acid turnover in exocrine pancreas and perhaps in membrane fusion events associated with exocytosis.     

Organellar proteomics to create the cell map

The elucidation of a complete, accurate, and permanent representation of the proteome of the mammalian cell may be achievable piecemeal by an organellar based approach. The small volume of organelles assures high protein concentrations. Providing isolated organelles are homogenous, this assures reliable protein characterization within the sensitivity and dynamic range limits of current mass spec based analysis. The stochastic aspect of peptide selection by tandem mass spectrometry for sequence determination by fragmentation is dealt with by multiple biological replicates as well as by prior protein separation on 1-D gels. Applications of this methodology to isolated synaptic vesicles, clathrin coated vesicles, endosomes, phagosomes, endoplasmic reticulum, and Golgi apparatus, as well as Golgi-derived COPI vesicles, have led to mechanistic insight into the identity and function of these organelles.     

Analysis of the association of syncollin with the membrane of the pancreatic zymogen granule

Syncollin is a pancreatic zymogen granule protein that was isolated through its ability to bind to syntaxin. Here we show that syncollin has a cleavable signal sequence and can be removed from granule membranes by washing with sodium carbonate. When membranes were subjected to Triton X-114 partitioning, syncollin was found predominantly in the aqueous phase, indicating that it is not sufficiently hydrophobic to be embedded in the membrane. Syncollin has intramolecular disulfide bonds and was accessible to water-soluble cross-linking and biotinylating reagents only when granules were lysed by sonication. These results indicate that syncollin is tightly bound to the luminal surface of the granule membrane. In situ, syncollin was resistant to proteases such as trypsin. When granule membranes were solubilized in ionic detergents such as deoxycholate, this trypsin resistance was maintained, and syncollin migrated on sucrose density gradients as a large (150 kDa) protein. In contrast, in non-ionic detergents such as Triton X-100, syncollin became partially sensitive to trypsin and behaved as a monomer. Syncollin in alkaline extracts of granule membranes was also monomeric. However, reduction of the pH regenerated the oligomeric form, which was insoluble. We conclude that syncollin exists as a homo-oligomer and that its ability to self-associate can be reversibly modulated via changes in pH. In light of our findings, we reassess the likely role of syncollin in the pancreatic acinar cell.     

Identification of the proteins exposed on the cytoplasmic surface of the pancreatic zymogen granule

Lactoperoxidase-catalyzed ¹²⁵I-iodination was used to label pancreatic zymogen granules. Membrane proteins facing the cytoplasmic surface were specifically labeled. Two low molecular weight proteins of 17000 and 15000 were intensely labeled at 0°C. Another small 13 kDa protein was strongly iodinated at 25°C along with some others, including the 29 kDa subunit of the ATP diphosphohydrolase. The major glycoprotein of the granule membrane was not iodinated but the presence of an iodinated 80 kDa protein suggests that proteolytic fragments of the 92 kDa glycoprotein were accessible to iodination on the intact granule. These proteins localized on the cytoplasmic surface of the granule are believed to play a major role in the exocytotic phenomenon of the exocrine pancreas.     

Organellar proteomics: the prizes and pitfalls of opening the nuclear envelope

Proteomic studies have the potential to comprehensively define the composition of organelles but are limited by the organellar cross-contamination that arises during subcellular fractionation. Comparative proteomics of organellar subfractions can mitigate these problems, as demonstrated by a recent study involving the nuclear envelope.     

Phosphorylation of a pancreatic zymogen granule membrane protein by endogenous calcium/phospholipid-dependent protein kinase

The occurrence of phospholipid-sensitive calcium-dependent protein kinase (referred to as C kinase) and its endogenous substrate proteins was examined in a membrane preparation from rat pancreatic zymogen granules. Using exogenous histone H1 as substrate, C kinase activity was found in the membrane fraction. The kinase was solubilized from membranes using Triton X-100 and partially purified using DEAE-cellulose chromatography. An endogenous membrane protein (Mr approximately equal to 18 000) was found to be specifically phosphorylated in the combined presence of Ca2+ and phosphatidylserine. Added diacylglycerol was effective in stimulating phosphorylation of exogenous histone by the partially purified C kinase, but had no effect upon phosphorylation of the endogenous 18 kDa protein by the membrane-associated C kinase. Phosphorylation of the 18 kDa protein was rapid (detectable within 30 s following exposure to Ca2+ and phosphatidylserine), and highly sensitive to Ca2+ (Ka = 4 microM in the presence of phosphatidylserine). These findings suggest a role for this Ca2+-dependent protein phosphorylation system in the regulation of pancreatic exocrine function.     

Elastic behavior of zymogen granule membranes in response to changes in pH and pCa.

In the process of secretion, the membrane of secretory granules is expected to change its elastic behavior. Elastic modulus of the membrane of zymogen granules, prepared from the rat pancreas acinar cell, was measured by an osmotic swelling method. The elastic modulus of the granule membrane at pCa 8 reduced from the maximal value of 230 dyn/cm at pH 6.0 to almost zero at pH 7.5. In a cytosol of an acinar cell, calcium ions play an important role as a second messenger in secretion. The elastic modulus of the granule membrane reduced in a sigmoidal fashion at pCa between 7.0 and 6.0. This range of pCa corresponds to a physiological rise of free Ca2+ concentrations in the cell cytosol when stimulated by external secretagogues. Reduction of the elastic modulus indicates that the state of the granule membrane switches to a more flexible one in which the granule is easy to appose to the cell plasma membrane and then swell as a final step of exocytosis.