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© The Rockefeller University Press, 0021-9525/1998//1331 $5.00
The Journal of Cell Biology, Volume 140, Number 6, , 1998 1331-1346

Article

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



Andreas Krömer, Michael M. Glombik, Wieland B. Huttner, and Hans-Hermann Gerdes

Department of Neurobiology, University of Heidelberg, 69120 Heidelberg, Germany

Sorting of regulated secretory proteins in the TGN to immature secretory granules (ISG) is thought to involve at least two steps: their selective aggregation and their interaction with membrane components destined 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. Upon transfection, i.e., with ongoing synthesis of endogenous granins, the sorting of the deletion mutant was only slightly impaired compared to full-length CgB. To investigate whether this sorting was due to coaggregation of the deletion mutant with endogenous granins, we expressed human CgB using recombinant vaccinia viruses, under conditions in which the synthesis of endogenous granins in the infected PC12 cells was shut off. In these conditions, {Delta}cys-hCgB, in contrast to full-length hCgB, was no longer sorted to ISG, but exited from the TGN in constitutive secretory vesicles. Coexpression of full-length hCgB together with {Delta}cys-hCgB by double infection, using the respective recombinant vaccinia viruses, rescued the sorting of the deletion mutant to ISG. In conclusion, our data show that (a) the disulfide-bonded loop is essential for sorting of CgB to ISG and (b) the lack of this structural motif can be compensated by coexpression of loop-bearing CgB. Furthermore, comparison of the two expression systems, transfection and vaccinia virus–mediated expression, reveals that analyses under conditions in which host cell secretory protein synthesis is blocked greatly facilitate the identification of sequence motifs required for sorting of regulated secretory proteins to secretory granules.

Abbreviations used in this paper: AT, {alpha}1-antitrypsin containing a tyrosine sulfation site; CgA, chromogranin A; CgB, chromogranin B; CV, constitutive secretory vesicles; EGFP, enhanced green fluorescent protein; gpt, guanine phosphoribosyltransferase; hCgB, human CgB; hsPG, heparansulfate proteoglycan; ISG, immature secretory granules; pfu, plaque-forming unit; PI, post infection; POMC, proopiomelanocorticotrophic hormone; rCgB, rat CgB; rSgII, rat SgII; SgII, secretogranin II; Seff, storage efficiency; vv, vaccinia virus.

W.B. Huttner and H.-H. Gerdes are recipients of grants from the Deutsche Forschungsgemeinschaft (SFB 317/C2 and C7) and the European Commission (Biotechnology ERBBIO4CT960058, TMR ERB-FMRX-CT96-0023).

Address all correspondence to Hans-Hermann Gerdes, Department of Neurobiology, University of Heidelberg, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany. Tel.: 49 6221 548317. Fax: 49 6221 548301. E-mail: hhgerdes{at}sun0.urz.uni-heidelberg.de

Andreas Krömer's present address is Institut für Klinische Pharmakologie Bobenheim, Prof. Dr. Lücker GmbH, Richard-Wagner-Straße 20, D-67269 Grünstadt, Germany.


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