caa a22 4500 475771397 CHVBK 20180406123618.0 cr unu---uuuuu 170329e20000901xx s 000 0 eng 10.1007/s001250051507 doi (NATIONALLICENCE)springer-10.1007/s001250051507 Trafficking of non-regulated secretory proteins in insulin secreting (INS-1) cells [Elektronische Daten] [M. Molinete, V. Lilla, R. Jain, P. B. M. Joyce, S.-U. Gorr, M. Ravazzola, P. A. Halban] Aims/hypothesis. 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. Conclusion/interpretation. 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. [Diabetologia (2000) 43: 1157-1164] Springer-Verlag Berlin Heidelberg, 2000 Keywords GFP nationallicence insulin nationallicence regulated secretory pathway nationallicence secretory granules nationallicence secretory alkaline phosphatase nationallicence Molinete M. Louis-Jeantet Research Laboratories, University Medical Centre, Geneva, Switzerland, CH aut Lilla V. Louis-Jeantet Research Laboratories, University Medical Centre, Geneva, Switzerland, CH aut Jain R. Department of Biological and Biophysical Sciences, University of Louisville School of Dentistry, Louisville, Kentucky, USA, US aut Joyce P. B. M. Department of Chemistry and Biochemistry and Centre for Structural and Functional Genomics, Concordia University, Montreal, Quebec, Canada, CA aut Gorr S.-U Department of Biological and Biophysical Sciences, University of Louisville School of Dentistry, Louisville, Kentucky, USA, US aut Ravazzola M. Department of Morphology, University Medical Centre, Geneva, Switzerland, CH aut Halban P. A. Louis-Jeantet Research Laboratories, University Medical Centre, Geneva, Switzerland, CH aut https://doi.org/10.1007/s001250051507 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s001250051507 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Molinete M. Louis-Jeantet Research Laboratories, University Medical Centre, Geneva, Switzerland, CH aut NATIONALLICENCE 700 1- Lilla V. Louis-Jeantet Research Laboratories, University Medical Centre, Geneva, Switzerland, CH aut NATIONALLICENCE 700 1- Jain R. Department of Biological and Biophysical Sciences, University of Louisville School of Dentistry, Louisville, Kentucky, USA, US aut NATIONALLICENCE 700 1- Joyce P. B. M. Department of Chemistry and Biochemistry and Centre for Structural and Functional Genomics, Concordia University, Montreal, Quebec, Canada, CA aut NATIONALLICENCE 700 1- Gorr S.-U Department of Biological and Biophysical Sciences, University of Louisville School of Dentistry, Louisville, Kentucky, USA, US aut NATIONALLICENCE 700 1- Ravazzola M. Department of Morphology, University Medical Centre, Geneva, Switzerland, CH aut NATIONALLICENCE 700 1- Halban P. A. Louis-Jeantet Research Laboratories, University Medical Centre, Geneva, Switzerland, CH aut Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer