Intracellular degradation of prohormone-chloramphenicol-acetyl-transferase chimeras in a pre-lysosomal compartment.
Small peptide hormones (less than 50 amino acids) are synthesized as larger inactive precursors. Work from several laboratories, including our own, has implicated the propeptide of various precursors in mediating intracellular transport and targeting to secretory granules. We previously demonstrated that the proregion of prosomatostatin, one of the simplest peptide hormone precursors, when fused to alpha-globin, enabled the globin polypeptide to be transported to the regulated secretory pathway. To identify sorting motifs in this propeptide, we have now constructed a chimera comprising the somatostatin signal peptide and proregion fused to chloramphenicol acetyl transferase (CAT) and a control protein consisting of the signal peptide fused to CAT, both of which were expressed in rat anterior-pituitary GH3 cells. Both molecules were translocated into the endoplasmic reticulum (ER) efficiently and core-glycosylated on the single cryptic N-linked glycosylation site present in CAT. Surprisingly, the glycosylated propeptide-CAT and signal without CAT were degraded intracellularly with half-lives of 30 min and 90 min, respectively. Based on the kinetics of degradation, temperature sensitivity, and resistance to lysosomotrophic agents, we suggest that degradation occurred in the ER. Our data imply that the pro-region is not an a priori universal sorter, but only directs heterologous peptides to the secretory pathway when the passenger peptide assumes a secretion-competent conformation.
Danoff
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Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461.
Mai
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Shields
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D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006031:Glycosylation; D008969:Molecular Sequence Data; D010903:Pituitary Gland, Anterior / Q000166:cytology / Q000378:metabolism*; D010957:Plasmids; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D013004:Somatostatin / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D011498:Protein Precursors; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D013004:Somatostatin; D015500:Chloramphenicol O-Acetyltransferase