Abstract

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 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.

Highlights

  • 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

  • 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

  • To distinguish intrinsic membrane proteins from peripheral and soluble proteins, a quantitative proteomic strategy was used to measure the relative abundances of ZG proteins during membrane purification

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Summary

Organellar Proteomics

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. To distinguish intrinsic membrane proteins from peripheral and soluble proteins, a quantitative proteomic strategy was used to measure the relative abundances of ZG proteins during membrane purification. June 3, 2005, and in revised form, October 31, 2005 Published, MCP Papers in Press, November 8, 2005, DOI 10.1074/ mcp.M500172-MCP200 1 The abbreviations used are: ZG, zymogen granule; iTRAQ, isobaric tag for relative and absolute quantification; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; TMHMM, Transmembrane Hidden Markov Model; VAMP, vesicle-associated membrane protein; 2D, two-dimensional; GPI, glycosylphosphatidylinositol; GE, gel electrophoresis; GTP␣S, guanosine 5Ј-O-(1-thiotriphosphate)

EXPERIMENTAL PROCEDURES
Pancreatic Zymogen Granule Membrane Proteins
RESULTS
Similar to osmotic stress protein
DISCUSSION
TM domainsa
Full Text
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