Insulin targeting to the regulated secretory pathway after fusion with green fluorescent protein and firefly...

We have prepared recombinant cDNAs encoding chimaeras between human preproinsulin (sp.B.C.A., for B-, Connecting- and A-peptides) and a thermostable mutant of green fluorescent protein (GFPS65T,V163A, GFP*). The subcellular localization of the expressed chimaeras was monitored in living insulin-secreting INS-1 beta-cells by laser scanning confocal microscopy. When GFP* was fused at the immediate N-terminus of the B-chain (sp.[GFP*].B.C.A.myc) two distinct patterns of fluorescence were apparent. In 1530/1740 cells examined, fluorescence was confined to a reticular, exclusively extranuclear structure, and closely co-localized with the endoplasmic reticulum marker, calreticulin. However, 210/1740 (12.1%) of cells displayed punctate fluorescence, which partially co-localized with the trans-Golgi network marker, TGN 38, and with the dense core secretory granule marker, phogrin. Since secretion of GFP* fluorescence into the medium could not readily be measured, we prepared a chimaera in which firefly luciferase was fused at the C-terminus of proinsulin (sp.B.C.A.myc.[Luc]). This chimaera displayed a distribution closely similar to that of sp.[GFP*].B.C.A. myc, but with a lower proportion (15/310, 4.8%) of the cells showing clear punctate distribution. At substimulatory glucose concentrations (3 mM) secretion of sp.B.C.A.myc.[Luc] could not be detected (rate of release into the medium identical with that of the cytosolic Renilla reniformis luciferase), indicating that the chimaera did not enter the constitutive secretory pathway. However, elevated (30 mM) glucose stimulated the release of the sp.B.C.A.myc. [Luc] luciferase chimaera, without a detectable effect on R. reniformis luciferase release. These data suggest that fusion of insulin, and the much larger photoproteins GFP* and luciferase, leads predominantly to misfolding and retention in the endoplasmic reticulum. However, the properly folded chimaeras are apparently still correctly targeted to the regulated, rather than the constitutive, secretory pathway. These chimaeras should therefore be valuable tools to monitor the exocytosis of insulin in real time.