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J. Cell Biol.,
Volume 140, Number 6, March 23, 1998 1331-1346
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 (
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, 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 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.
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