Analysis of low abundance membrane-associated proteins from rat pancreatic zymogen granules.
Zymogen granules (ZG) are specialized storage organelles in the exocrine pancreas that allow the sorting, packaging, and regulated apical secretion of digestive enzymes. As there is a critical need for further understanding of the key processes in regulated secretion to develop new therapeutic options in medicine, we applied a suborganellar proteomics approach to identify peripheral membrane-associated ZG proteins. We focused on the analysis of a "basic" group (pH range 6.2-11) with about 46 spots among which 44 were identified by tandem mass spectrometry. These spots corresponded to 16 unique proteins, including rat mast cell chymase (RMCP-1) and peptidyl-prolyl cis-trans isomerase B (PpiB; cyclophilin B), an ER-resident protein. To confirm that these proteins were specific to zymogen granules and not contaminants of the preparation, we conducted a series of validation experiments. Immunoblotting of ZG subfractions revealed that chymase and PpiB behaved like bona fide peripheral membrane proteins. Their expression in rat pancreas was regulated by feeding behavior. Ultrastructural and immunofluorescence studies confirmed their ZG localization. Furthermore, a chymase-YFP fusion protein was properly targeted to ZG in pancreatic AR42J cells. Interestingly, for both proteins, proteoglycan-binding properties have been reported. The importance of our findings for sorting and packaging during ZG formation is discussed.
Borta
Heike
H
Department of Cell Biology and Cell Pathology, Philipps University of Marburg, Robert Koch Strasse 6, Marburg, Germany.
Aroso
Miguel
M
Rinn
Cornelia
C
Gomez-Lazaro
Maria
M
Vitorino
Rui
R
Zeuschner
Dagmar
D
Grabenbauer
Markus
M
Amado
Francisco
F
Schrader
Michael
M
D000818:Animals; D045744:Cell Line, Tumor; D053818:Chymases / Q000235:genetics / Q000378:metabolism; D021983:Cyclophilins / Q000235:genetics / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D046790:Pancreas, Exocrine / Q000378:metabolism* / Q000648:ultrastructure; D040901:Proteomics / Q000379:methods*; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D022142:Secretory Vesicles / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
D008565:Membrane Proteins; C081786:cyclophilin B; D053818:Chymases; D021983:Cyclophilins