ESSENTIAL ROLE OF THE DISULFIDE-BONDED LOOP OF CHROMOGRANIN-B FOR SORTING TO SECRETORY GRANULES IS REVEALED BY EXPRESSION OF A DELETION MUTANT IN THE ABSENCE OF ENDOGENOUS GRANIN SYNTHESIS

Sorting of regulated secretory proteins in the TGN to immature secreto ry granules (ISG) is thought to involve at least two steps: their sele ctive aggregation and their interaction with membrane components desti ned to ISG, Here, we have investigated the sorting of chromogranin B ( CgB), a member of the granin family present in the secretory granules of many endocrine cells and neurons, Specifically, we have studied the role of a candidate structural motif implicated in the sorting of CgB , the highly conserved NH2-terminal disulfide-bonded loop. Sorting to ISG of full-length human CgB and a deletion mutant of human CgB (Delta cys-hCgB) lacking the 22-amino acid residues comprising the disulfide -bonded loop was compared in the rat neuroendocrine cell line PC12. Up on transfection, i.e., with ongoing synthesis of endogenous granins, t he sorting of the deletion mutant was only slightly impaired compared to full-length CgB, To investigate whether this sorting was due to coa ggregation of the deletion mutant with endogenous granins, we expresse d human CgB using recombinant vaccinia viruses, under conditions in wh ich the synthesis of endogenous granins in the infected PC12 cells was shut off, In these conditions, Delta cys-hCgB, in contrast to full-le ngth hCgB, was no longer sorted to ISG, but exited from the TGN in con stitutive secretory vesicles, Coexpression of full-length hCgB togethe r with Delta cys-hCgB by double infection, using the respective recomb inant vaccinia viruses, rescued the sorting of the deletion mutant to ISG, In conclusion, our data show that (a) the disulfide-bonded loop i s essential for sorting of CgB to ISG and (b) the lack of this structu ral motif can be compensated by coexpression of loop-bearing CgB, Furt hermore, comparison of the two expression systems, transfection and va ccinia virus-mediated expression, reveals that analyses under conditio ns in which host cell secretory protein synthesis is blocked greatly f acilitate the identification of sequence motifs required for sorting o f regulated secretory proteins to secretory granules.