A novel zymogen granule protein (ZG29p) and the nuclear protein MTA1p are differentially expressed by alternative transcription initiation in pancreatic acinar cells of the rat.
Using a polyclonal antibody against purified zymogen granule membrane components from rat pancreas a cDNA coding for the 29 kDa protein (ZG29p) was identified by immunoscreening of a hormonally stimulated pancreas cDNA library. Western blot analysis suggests that ZG29p is a pancreas-specific protein and immunofluorescence shows that ZG29p is mainly associated with zymogen granules. Analysis of subcellular fraction applying immunoblotting revealed that ZG29p was localized mainly in the soluble fraction of zymogen granules and in a Golgi- and RER-enriched fraction, but was absent from the cytosol. In isolated zymogen granule content ZG29p was associated with protein complexes containing amylase as main constituent. The cDNA coding for ZG29p is homologous to the C-terminal region of the candidate metastasis-associated gene mta1. Northern blot analysis and RT-PCR showed that no MTA1 mRNA is present in pancreas from fasted rats and in the rat pancreas carcinoma cell line AR4-2J in its protodifferentiated state. Although no ZG29p specific mRNA was seen in the northern blot analysis, RT-PCR showed that ZG29p was expressed under both non-stimulated and stimulated conditions. The expression of MTA1 was up-regulated in the pancreas by endogenous cholecystokinin release and in AR4-2J after induction of cellular differentiation by dexamethasone. Western blotting and immunofluorescense studies indicated that MTA1p is localized in the nucleus in all tissues studied. Using genomic DNA in PCR analysis it was shown that two short introns are present flanking the sequences of the 5'end of ZG29p cDNA. One intron contains consensus elements required for pancreas specific transcription initiation, suggesting that MTA1 and ZG29 are differentially expressed by alternative transcription initiation in the pancreas. The localisation of MTA1p in the nucleus of most cell types could signify a general role in gene regulation, while the cell type specific and exclusive expression of ZG29p in pancreatic acinar cells could indicate a role in granule formation.
Kleene
R
R
Department of Cell Biology and Cell Pathology, Philipps University, Marburg/Germany. kleene@mailer.uni-marburg.de
Zdzieblo
J
J
Wege
K
K
Kern
H F
HF
D000595:Amino Acid Sequence; D000681:Amylases / Q000378:metabolism; D000818:Animals; D002458:Cell Fractionation; D003001:Cloning, Molecular; D003594:Cytoplasmic Granules / Q000378:metabolism* / Q000648:ultrastructure*; D018870:Endoplasmic Reticulum, Rough / Q000378:metabolism / Q000648:ultrastructure; D005260:Female; D005786:Gene Expression Regulation; D006056:Golgi Apparatus / Q000378:metabolism / Q000648:ultrastructure; D006655:Histone Deacetylases; D008297:Male; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics; D009928:Organ Specificity; D010179:Pancreas / Q000166:cytology* / Q000378:metabolism*; D014176:Protein Biosynthesis; D011506:Proteins / Q000737:chemistry / Q000235:genetics*; D051381:Rats; D017208:Rats, Wistar; D012097:Repressor Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015534:Trans-Activators; D014158:Transcription, Genetic
C089320:MTA1 protein, human; C121683:Mta1 protein, rat; D009687:Nuclear Proteins; D011506:Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D000681:Amylases; D006655:Histone Deacetylases