Global Topology Analysis of Pancreatic Zymogen Granule Membrane Proteins*□S

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 cytoplasmand 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. Molecular & Cellular Proteomics 7:2323–2336, 2008. The acinar cells of the exocrine pancreas are the functional units of digestive enzyme synthesis, storage, and secretion. Digestive enzymes are stored in large vesicles within these cells known as zymogen granules (ZGs). The ZG is the secretory organelle responsible for transport, storage, and secretion of digestive enzymes and has long been a model for understanding secretory granule functions (1, 2). It is believed that the ZG membrane (ZGM) carries at least part of the molecular machinery responsible for digestive enzyme sorting, granule trafficking, and exocytosis. Therefore elucidating the ZGM molecular architecture is critical for studying ZG function. The overall goal of our studies is to build a quantitative, architectural model of the ZGM that will direct new hypotheses for subsequent functional analysis of this prototypic secretory granule. This model will ultimately comprise not only the complete protein components of the ZGM but also their membrane topologies, absolute quantities, and protein complexes with which they are associated. As the first step toward this goal, we recently conducted the first comprehensive proteomics analysis of ZGM and identified over 100 proteins (3). The next question to be addressed is how these proteins are organized across the ZGM, namely the membrane topology of ZGM proteins. The topological organization of a ZGM protein relative to the lipid bilayer dictates its accessibility to interacting partners and modifying enzymes. Therefore, an accurate topology model describing the number of transmembrane spans and the orientation of a ZGM protein is essential for understanding its correct function. This importance is highlighted in the case of syncollin that was originally suggested as a Ca -sensitive regulator of the SNARE complex but then identified as a luminal peripheral membrane protein likely playing a role in ZG maturation (4). Despite the importance of membrane topol-