Trafficking of non-regulated secretory proteins in insulin secreting (INS-1) cells.
OBJECTIVE
Sorting of proinsulin to the regulated secretory pathway of pancreatic beta cells and retention of insulin in dense-core granules of this pathway is remarkably efficient. To monitor the specificity of these events, the secretion of two exogenous secretory proteins not known to carry information for sorting or retention in the regulated pathway was investigated in INS-1 cells.


METHODS
SEGFP, a fusion protein consisting of a signal peptide N-terminal to EGFP (mutant green fluorescent protein with enhanced fluorescence) and secreted alkaline phosphatase (SEAP) were expressed in INS-1 cells by transfection and by infection with recombinant adenovirus, respectively. Secretion of SEGFP was monitored by quantitative western blotting and that of SEAP by its activity.


RESULTS
Secreted alkaline phosphatase showed high basal secretion (6.6% total) but only modest (3.6-fold) stimulation of secretion by secretagogues, in keeping with secretion largely through the constitutive pathway. By contrast SEGFP had a secretory pattern similar to insulin, with low basal secretion (0.8% total) and 16-fold stimulation by secretagogues. Granular localization of SEGFP was confirmed by high resolution electron microscopy immunocytochemistry. Pulse-chase experiments indicated retention of SEGFP in granules at least 24 h after synthesis. The secretory SEGFP, but not cytosolic EGFP, formed disulphide-linked oligomers. This could be implicated in its regulated secretion.


CONCLUSIONS
These data indicate that in INS-1 cells SEGFP, but not SEAP, is unexpectedly handled as a regulated secretory protein and stored along with insulin in granules. This raises questions about the specificity and mechanism of the sorting of proteins to granules in INS-1 cells or their retention therein or both.
Molinete
M
M
Louis-Jeantet Research Laboratories, University Medical Centre, Geneva, Switzerland.
Lilla
V
V
Jain
R
R
Joyce
P B
PB
Gorr
S U
SU
Ravazzola
M
M
Halban
P A
PA
D000256:Adenoviridae; D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D003594:Cytoplasmic Granules / Q000378:metabolism / Q000648:ultrastructure; D003600:Cytosol / Q000378:metabolism / Q000648:ultrastructure; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000378:metabolism / Q000502:physiology*; D007700:Kinetics; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
D007328:Insulin; D008164:Luminescent Proteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D000469:Alkaline Phosphatase