INSULIN TARGETING TO THE REGULATED SECRETORY PATHWAY AFTER FUSION WITH GREEN FLUORESCENT PROTEIN AND FIREFLY LUCIFERASE

We have prepared recombinant cDNAs encoding chimaeras between human pr eproinsulin (sp.B.C.A, for B-, Connecting- and A-peptides) and a therm ostable mutant of green fluorescent protein (GFP(S65T,V163A), GFP). T he subcellular localization of the expressed chimaeras was monitored i n 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 wer e apparent. In 1530/1740 cells examined, fluorescence was confined to a reticular, exclusively extranuclear structure, and closely co-locali zed 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 th e dense core secretory granule marker, phogrin. Since secretion of GFP fluorescence into the medium could not readily be measured, we prepa red a chimaera in which firefly luciferase was fused at the C-terminus of proinsulin (sp.B.C.A.myc.[Luc]). This chimaera displayed a distrib ution 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 distri bution. At substimulatory glucose concentrations (3 mM) secretion of s p.B.C.A.myc.[Luc] could not be detected (rate of release into the medi um identical with that of the cytosolic Renilla renifol mis luciferase ), indicating that the chimaera did not enter the constitutive secreto ry pathway. However, elevated (30 mM) glucose stimulated the release o f the sp.B.C.A.myc.[Luc] luciferase chimaera, without a detectable eff ect on R. reniformis luciferase release. These data suggest that fusio n of insulin, and the much larger photoproteins GFP and luciferase, l eads predominantly to misfolding and retention in the endoplasmic reti culum. However, the properly folded chimaeras are apparently still cor rectly targeted to the regulated, rather than the constitutive, secret ory pathway. These chimaeras should therefore be valuable tools to mon itor the exocytosis of insulin in real time.