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Association of nucleoside diphosphate kinase with pancreatic zymogen granules: effects of local GTP generation on granule membrane characteristics.

ABSTRACT: It is well established that both GTP-binding proteins and phosphoproteins are involved in the control of exocytosis in the exocrine pancreas. Exocytotic membrane fusion is stimulated by guanosine 5'-[gamma-thio]triphosphate, and the phosphorylation states of several proteins, including at least one on the zymogen granule membrane, are known to change during exocytosis. We show here that a nucleoside diphosphate kinase is associated with the cytoplasmic face of pancreatic zymogen granules. This enzyme behaves as a phosphoprotein of apparent molecular mass 21 kDa on SDS/polyacrylamide gels, and is able to produce GTP by using ATP to phosphorylate endogenous GDP. GTP production by nucleoside diphosphate kinase is stimulated by the wasp venom peptide mastoparan, both through a direct action on the enzyme and through its ability to increase the availability of endogenous GDP. Two effects of the GTP produced by nucleoside diphosphate kinase are demonstrated: phosphorylation of a 37 kDa zymogen granule protein on histidine residues, and stimulation of the fusion of zymogen granules with pancreatic plasma membranes in vitro. These results suggest that granule-associated nucleoside diphosphate kinase is able to maintain local GTP concentrations, and raise the possibility that it might be involved in the control of exocytosis in the pancreatic acinar cell.

SUBMITTER: Marciniak SJ

PROVIDER: S-EPMC1217356 | biostudies-other | 1996 May

REPOSITORIES: biostudies-other

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Project description:The zymogen granule is the specialized organelle in pancreatic acinar cells for digestive enzyme storage and regulated secretion and is a classic model for studying secretory granule function. Our long term goal is to develop a comprehensive architectural model for zymogen granule membrane (ZGM) proteins that would direct new hypotheses for subsequent functional studies. Our initial proteomics analysis focused on identification of proteins from purified ZGM (Chen, X., Walker, A. K., Strahler, J. R., Simon, E. S., Tomanicek-Volk, S. L., Nelson, B. B., Hurley, M. C., Ernst, S. A., Williams, J. A., and Andrews, P. C. (2006) Organellar proteomics: analysis of pancreatic zymogen granule membranes. Mol. Cell. Proteomics 5, 306-312). In the current study, a new global topology analysis of ZGM proteins is described that applies isotope enrichment methods to a protease protection protocol. Our results showed that tryptic peptides of ZGM proteins were separated into two distinct clusters according to their isobaric tag for relative and absolute quantification (iTRAQ) ratios for proteinase K-treated versus control zymogen granules. The low iTRAQ ratio cluster included cytoplasm-orientated membrane and membrane-associated proteins including myosin V, vesicle-associated membrane proteins, syntaxins, and all the Rab proteins. The second cluster having unchanged ratios included predominantly luminal proteins. Because quantification is at the peptide level, this technique is also capable of mapping both cytoplasm- and lumen-orientated domains from the same transmembrane protein. To more accurately assign the topology, we developed a statistical mixture model to provide probabilities for identified peptides to be cytoplasmic or luminal based on their iTRAQ ratios. By implementing this approach to global topology analysis of ZGM proteins, we report here an experimentally constrained, comprehensive topology model of identified zymogen granule membrane proteins. This model contributes to a firm foundation for developing a higher order architecture model of the ZGM and for future functional studies of individual ZGM proteins.

Project description:The regulation of Cl- conductance by cytoplasmic nucleotides was investigated in pancreatic and parotid zymogen granules. Cl- conductance was assayed by measuring the rate of cation-ionophore-induced osmotic lysis of granules suspended in iso-osmotic salt solutions. Both inhibition and stimulation were observed, depending on the type and concentration of nucleotide. Under optimal conditions, the average inhibition measured in different preparations was 1.6-fold, whereas the average stimulation was 4.4-fold. ATP was inhibitory at 1-10 microM but stimulated Cl- conductance above 50 microM. Stimulation by ATP was more pronounced in granules with low endogenous Cl- conductance. The potency of nucleotides in terms of inhibition was ATP greater than adenosine 5'-[gamma-thio]triphosphate (ATP[S]) greater than UTP much greater than or equal to CTP much greater than or equal to GTP much greater than or equal to guanosine 5'-[gamma-thio]triphosphate (GTP[S]) much greater than or equal to ITP. The potency with respect to stimulation had the following order: adenosine 5'-[beta gamma-methylene]triphosphate (App[CH2]p) greater than ATP greater than guanosine 5'-[beta-thio]diphosphate (GDP[S]). Adenosine 5'-[beta gamma-imido]triphosphate (App[NH]p) was also stimulatory, and was more potent than ATP in the parotid granules, but less potent in the pancreatic granules. Aluminium fluoride stimulated Cl- conductance maximally at 15-30 microM-Al3+ and 10-15 mM-F. F was less effective at higher concentrations. Protein phosphorylation by kinases was apparently not involved, since the nucleotide effects (1) could be mimicked by non-hydrolysable analogues of ATP and GTP, (2) showed reversibility, and (3) were not abolished by the protein kinase inhibitors 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H-7) or staurosporine. The data suggest the presence of at least two binding sites for nucleotides, whereby occupancy of one induces inhibition and occupancy of the other induces stimulation.

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Association of nucleoside diphosphate kinase with pancreatic zymogen granules: effects of local GTP generation on granule membrane characteristics.

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