Conversion of proinsulin to insulin occurs coordinately with acidification of maturing secretory vesicles

Orci, Lelio; Ravazzola, M.; Amherdt, M.; Madsen, O.; Perrelet, Alain; Vassalli, Jean-Dominique; Anderson, R. G.

doi:10.1083/jcb.103.6.2273

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Title		Conversion of proinsulin to insulin occurs coordinately with acidification of maturing secretory vesicles
Authors		Orci, Lelio Ravazzola, M. Amherdt, M. Madsen, O. Perrelet, Alain Vassalli, Jean-Dominique Anderson, R. G.
Published in		The Journal of Cell Biology. 1986, vol. 103, no. 6 Pt 1, p. 2273-2281
Abstract		Proinsulin is a single polypeptide chain composed of the B and A subunits of insulin joined by the C-peptide region. Proinsulin is converted to insulin during the maturation of secretory vesicles by the action of two proteases and conversion is inhibited by ionophores that disrupted intracellular H+ gradients. To determine if conversion of prohormone to hormone actually occurs in an acidic secretory vesicle, cultured rat islet cells were incubated in the presence of 3-(2,4-dinitroanilino)-3' amino-N-methyldipropylamine (DAMP), a basic congener of dinitrophenol that concentrates in acidic compartments and is retained there after aldehyde fixation. The cells were processed for indirect protein A-gold colocalization of DAMP, using a monoclonal antibody to dinitrophenol, and proinsulin, using a monoclonal antibody that exclusively reacts with the prohormone. The average density of DAMP-specific gold particles in immature secretory vesicles that contained proinsulin was 71/micron 2 (18 times cytoplasmic background), which indicated that this compartment was acidic. However, the density of DAMP-specific gold particles in the insulin-rich mature secretory vesicle averaged 433/micron 2. This suggests that although proinsulin conversion occurs in an acidic compartment, the secretory vesicles become more acidic as they mature. Since the concentration of anti-proinsulin IgG binding in secretory vesicles is inversely proportional to the conversion of proinsulin to insulin, we were able to determine that maturing secretory vesicles had to reach a critical pH before proinsulin conversion occurred.
Keywords		Animals — Antibodies, Monoclonal — Cytoplasmic Granules/ metabolism/ultrastructure — Fluorescent Antibody Technique — Insulin/analysis/ genetics — Islets of Langerhans/ metabolism/ultrastructure — Microscopy, Electron — Peptide Hydrolases/ metabolism — Proinsulin/analysis/ genetics — Protein Processing, Post-Translational — Rats — Rats, Inbred Strains
Identifiers		DOI: 10.1083/jcb.103.6.2273 PMID: 3536964
Full text		Article - Limited access to UNIGE Other version: http://jcb.rupress.org/content/103/6/2273.full.pdf
Structures		Faculté de médecine / Section de médecine clinique / Département de médecine
Citation (ISO format)		ORCI, Lelio et al. Conversion of proinsulin to insulin occurs coordinately with acidification of maturing secretory vesicles. In: The Journal of Cell Biology, 1986, vol. 103, n° 6 Pt 1, p. 2273-2281. doi: 10.1083/jcb.103.6.2273 https://archive-ouverte.unige.ch/unige:8947