Trafficking of mutant carboxypeptidase E to secretory granules in a beta-cell line derived from Cpe(fat)/Cpe(fat) mice.
We have reinvestigated the stability and intracellular routing of mutant carboxypeptidase E in NIT3 cells, a pancreatic beta-cell line derived from the Cpe(fat)/Cpe(fat) mouse. Pulse-chase experiments demonstrated that this protein has a half-life of approximately 3 h in these cells and that up to 45% of the proCPE(202) can escape degradation by the proteosome. In double-label immunofluorescence microscopy, a portion of the mutant CPE did not colocalize with calnexin, an endoplasmic reticulum marker, but was found in prohormone convertase 2-containing secretory granules, demonstrating that it had escaped degradation and arrived at a post-Golgi compartment. The mutant CPE as well as prohormone convertase 2 were secreted into the medium in a stimulated manner by treatment with the physiological secretagogue, glucagon-like peptide-1, consistent with its presence in granules of the regulated secretory pathway. The presence of mutant carboxypeptidase E in granules supports a potential role for its involvement as a sorting/retention receptor in the trafficking of proinsulin to the regulated secretory pathway.
Cawley
Niamh X
NX
Section on Cellular Neurobiology, Laboratory of Developmental Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
Rodriguez
Yazmin M
YM
Maldonado
Alex
A
Loh
Y Peng
YP
D000818:Animals; D015153:Blotting, Western; D043423:Carboxypeptidase H; D002268:Carboxypeptidases / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D002460:Cell Line; D004792:Enzyme Precursors / Q000032:analysis; D004795:Enzyme Stability; D019084:Fluorescent Antibody Technique, Indirect; D005934:Glucagon / Q000494:pharmacology; D052216:Glucagon-Like Peptide 1; D006207:Half-Life; D007515:Islets of Langerhans / Q000201:enzymology / Q000648:ultrastructure*; D051379:Mice; D008856:Microscopy, Fluorescence; D009154:Mutation; D010446:Peptide Fragments / Q000494:pharmacology; D045706:Proprotein Convertase 2; D011498:Protein Precursors / Q000494:pharmacology; D022142:Secretory Vesicles / Q000201:enzymology*; D013381:Subtilisins / Q000032:analysis / Q000378:metabolism
D004792:Enzyme Precursors; D010446:Peptide Fragments; D011498:Protein Precursors; D052216:Glucagon-Like Peptide 1; D005934:Glucagon; D002268:Carboxypeptidases; D043423:Carboxypeptidase H; D013381:Subtilisins; D045706:Proprotein Convertase 2