The zymogen granule (ZG) is the specialized organelle in pancreatic acinar cells for digestive enzyme storage and regulated secretion and has been a model for studying secretory granule functions. In an initial effort to comprehensively understand the functions of this organelle, we conducted a proteomic study to identify proteins from highly purified ZG membranes. By combining two-dimensional gel electrophoresis and two-dimensional LC with tandem mass spectrometry, 101 proteins were identified from purified ZG membranes including 28 known ZG proteins and 73 previously unknown proteins, including SNAP29, Rab27B, Rab11A, Rab6, Rap1, and myosin Vc. Moreover several hypothetical proteins were identified that represent potential novel proteins. The ZG localization of nine of these proteins was further confirmed by immunocytochemistry. To distinguish intrinsic membrane proteins from soluble and peripheral membrane proteins, a quantitative proteomic strategy was used to measure the enrichment of intrinsic membrane proteins through the purification process. The iTRAQ TM ratios correlated well with known or Transmembrane Hidden Markov Model-predicted soluble or membrane proteins. By combining subcellular fractionation with high resolution separation and comprehensive identification of proteins, we have begun to elucidate zymogen granule functions through proteomic and subsequent functional analysis of its membrane components. Molecular & Cellular Proteomics 5:306 -312, 2006.Pancreatic acinar cells are the functional units of digestive enzyme synthesis, storage, and secretion and have been a classical model for studying regulated exocytosis (1, 2). In acinar cells digestive enzymes are stored in a specialized organelle, the zymogen granule (ZG).1 Stimulation of acinar cells by secretagogues triggers fusion of ZG membranes with the apical membrane and the subsequent release of digestive enzymes. Early studies using SDS-PAGE indicated a relatively simple protein structure for the ZG membrane. Several more recent studies using 2D GE led to the identification of two additional major ZG membrane components, GP3 (3) and membrane dipeptidase (4). In another study, 14 spots were identified as small GTPases by [ 35 S]GTP␣S overlay on a 2D gel of ZG membrane proteins, but the identities of the spots remained unknown (5). In addition, a number of low abundance proteins, including Rab3D and several SNARE proteins, have been identified on ZG membranes by immunoblotting and immunocytochemistry (2). Despite its importance, a comprehensive proteomic analysis of the membrane protein components of ZGs has not been achieved.By combining 2D GE and 2D LC with tandem mass spectrometry, we report the identification of 101 proteins on ZG membranes, many of which had not been localized previously on ZGs including multiple small GTP-binding proteins, SNARE proteins, and molecular motor proteins. In addition, to distinguish intrinsic membrane proteins from peripheral and soluble proteins, a quantitative proteomic strategy was used to measure the r...

Methionine (Met) S-methyltransferase (MMT) catalyzes the synthesis of S-methyl-Met (SMM) from Met and S-adenosyl-Met(Ado-Met). SMM can be reconverted to Met by donating a methyl group to homocysteine (homo-Cys), and concurrent operation of this reaction and that mediated by MMT sets up the SMM cycle. SMM has been hypothesized to be essential as a methyl donor or as a transport form of sulfur, and the SMM cycle has been hypothesized to guard against depletion of the free Met pool by excess Ado-Met synthesis or to regulate Ado-Met level and hence the Ado-Met to S-adenosylhomo-Cys ratio (the methylation ratio). To test these hypotheses, we isolated insertional mmt mutants of Arabidopsis and maize (Zea mays). Both mutants lacked the capacity to produce SMM and thus had no SMM cycle. They nevertheless grew and reproduced normally, and the seeds of the Arabidopsis mutant had normal sulfur contents. These findings rule out an indispensable role for SMM as a methyl donor or in sulfur transport. The Arabidopsis mutant had significantly higher Ado-Met and lower S-adenosylhomo-Cys levels than the wild type and consequently had a higher methylation ratio (13.8 versus 9.5). Free Met and thiol pools were unaltered in this mutant, although there were moderate decreases (of 30%-60%) in free serine, threonine, proline, and other amino acids. These data indicate that the SMM cycle contributes to regulation of Ado-Met levels rather than preventing depletion of free Met.S-Methyl-Met (SMM) synthesis is a unique feature of plant sulfur and one-carbon metabolism (Pokorny et al., 1970; Mudd and Datko, 1990; Ranocha et al., 2001). SMM is formed by the S-adenosyl-Met (AdoMet)-dependent methylation of Met, catalyzed by Met S-methyltransferase (MMT; Bourgis et al., 1999). SMM can be reconverted to Met by transferring a methyl group to homo-Cys in a reaction mediated by homo-Cys S-methyltransferase (HMT; Ranocha et al., 2000). The tandem action of MMT and HMT, together with that of Ado-Met synthetase and S-adenoyslhomo-Cys (AdoHcy) hydrolase, sets up a futile cycle (the SMM cycle) in which Met is converted to SMM, and SMM is reconverted to Met (Mudd and Datko, 1990). This cycle in effect short-circuits the activated methyl cycle (Fig. 1), and each of its turns hydrolyzes a molecule of ATP to adenosine, pyrophosphate, and phosphate. The SMM cycle operates throughout the plant, and consumes one-half the Ado-Met produced in Arabidopsis leaves (Ranocha et al., 2001).The functions of SMM and its seemingly wasteful cycle are for the most part unknown. The only established role of SMM is in transporting reduced sulfur in the phloem, for which there is qualitative evidence in a range of plants including Arabidopsis (Bourgis et al., 1999 Article, publication date, and citation information can be found at www.plantphysiol.org/cgi

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. 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).1 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...

Improvements to phosphopeptide enrichment protocols employing titanium dioxide (TiO2) are described and applied to identification of phosphorylation sites on recombinant human cyclin-dependent kinase 2 (CDK2). Titanium dioxide binds phosphopeptides under acidic conditions, and they can be eluted under basic conditions. However, some nonphosphorylated peptides, particularly acidic peptides, bind and elute under these conditions as well. These nonphosphorylated peptides contribute significantly to ion suppression of phosphopeptides and also increase sample complexity. We show here that the conversion of peptide carboxylates to their corresponding methyl esters sharply reduces nonspecific binding, improving the selectivity for phosphopeptides, just as has been reported for immobilized metal affinity chromatography (IMAC) columns. We also present evidence that monophosphorylated peptides can be effectively fractionated from multiply phosphorylated peptides, as well as acidic peptides, via stepwise elution from TiO2 using pH step gradients from pH 8.5 to pH 11.5. These approaches were applied to human CDK2 phosphorylated in vitro by yeast CAK1p in the absence of cyclin. We confirmed phosphorylation at T160, a site previously documented and shown to be necessary for CDK2 activity. However, we also discovered several novel sites of partial phosphorylation at S46, T47, T165, and Y168 when ion-suppressing nonphosphorylated peptides were eliminated using the new protocols.

During mitosis, the stacked structure of the Golgi undergoes a continuous fragmentation process. The generated mitotic fragments are evenly distributed into the daughter cells and reassembled into new Golgi stacks. This disassembly and reassembly process is critical for Golgi biogenesis during cell division, but the underlying molecular mechanism is poorly understood. In this study, we have recapitulated this process using an in vitro assay and analyzed the proteins associated with interphase and mitotic Golgi membranes using a proteomic approach. Incubation of purified rat liver Golgi membranes with mitotic HeLa cell cytosol led to fragmentation of the membranes; subsequent treatment of these membranes with interphase cytosol allowed the reassembly of the Golgi fragments into new Golgi stacks. These membranes were then used for quantitative proteomics analyses by combining the isobaric tags for relative and absolute quantification approach with OFFGEL isoelectric focusing separation and liquid chromatography-matrix assisted laser desorption ionization-tandem mass spectrometry. In three independent experiments, a total of 1,193 Golgi-associated proteins were identified and quantified. These included broad functional categories, such as Golgi structural proteins, Golgi resident enzymes, SNAREs, Rab GTPases, cargo, and cytoskeletal proteins. More importantly, the combination of the quantitative approach with Western blotting allowed us to unveil 84 proteins with significant changes in abundance under the mitotic condition compared with the interphase condition. Among these proteins, several COPI coatomer subunits (␣, ␤, ␥, and ␦) are of particular interest. Altogether, this systematic quantitative proteomic study revealed candidate proteins of the molecular machinery that control the Golgi disassembly and reassembly processes in the cell cycle.The Golgi complex is the central organelle in the secretory pathway, essential for post-translational modifications, sorting, and trafficking of newly synthesized secretory and membrane proteins and lipids in all eukaryotic cells. The unique feature of the Golgi in almost all eukaryotic cells is the densely packed stacks of the flattened cisternal membranes. Processing enzymes in the Golgi complex, including those involved in modifying bound oligosaccharides, are arranged across the stack in the cis to trans order in which they function. In animal cells, stacks are often interconnected to form a ribbon-like structure that is localized adjacent to the nucleus. Despite its complicated morphology and function,