pH-independent and -dependent cleavage of proinsulin in the same secretory vesicle.

Abstract

Abstract. By quantitative immunoelectron microscopy and HPLC, we have studied the effect of disrupting pH gradients, by ammonium chloride, on proinsulin conversion in the insulin-producing B-cells of the islets of Langerhans. Proinsulin content and pH in single secretory vesicles were measured on consecutive serial sections immunostained alternately with antiproinsulin or anti-dinitrophenol (to reveal the pHsensitive probe DAMP) antibodies. Radioactively labeled proinsulin, proinsulin cleavage intermediates, and insulin were quantitated by HPLC analysis of extracts of islets treated in the same conditions. Cleavage at the C-peptide/A-chain junction is significantly less sensitive to pH gradient disruption than that of the B-chain/C-peptide junction, but the range of pH and proinsulin content in individual vesicles indicate that both cleavages occur in the same vesicle released from the TGN.IDIFICATIOr~ of many of the intracellular membrane bounded compartments that are created on the endocytotic and secretory pathways is coupled to orderly protein processing and membrane traffic. For example, during receptor-mediated endocytosis, low pH promotes the dissociation of the ligand from its receptor before fusion of the endosome with the lysosome. Acidification of vesicles originating from the TGN activates proteolytic enzymes that generate functional hormones by cleaving precursor proteins (e.g., prohormones) as they emerge from the Golgi (for review see Anderson and Orci, 1986;Mellman et al., 1986).Acidification is mediated by a membrane (H+)-ATPase that is able to maintain variable concentrations of protons within the vesicle lumen. As a consequence, the pH of sequential compartments in an endocytic or secretory pathway can change from 7.0 to as low as 4.5 (for review see AIAwqati, 1986). There is also clear evidence that in certain cells the pH of a compartment is tightly regulated (Mallya et al., 1992). Therefore, pH is often used to modulate chemical reactions that take place within intracellular vesicles.In the insulin-producing B-cell of the islet of Langerhans, biochemical and immunocytochemical studies have shown that the synthesis of proinsulin in the endoplasmic reticulum and its transport to the TGN occur in a relatively neutral pH environment. The conversion of proinsulin to insulin, however, is initiated in acidic (~pH 6.0) clathrin-coated secre-

A Novel GLP1 Receptor Interacting Protein ATP6ap2 Regulates Insulin Secretion in Pancreatic Beta Cells

“…Previous studies have shown the involvement of acidic secretory vesicles in insulin processing and maturation (66). Specifically, proprotein convertases 3 (PC1/3) and 2 (PC2) responsible for insulin processing from proinsulin are strictly pH-dependent (51,67). In line with this, the gene expression of insulin-processing enzymes PC1/3, PC2, and CPE were not changed in ATP6ap2 knockdown cells.…”

Proinsulin Is Refractory to Protein Fibrillation

“…Proteins were made 60 M in each of the following conditions: (i) degassed phosphate-buffered saline (PBS, 10 mM phosphate, 140 mM NaCl) at pH 7.4 (mimicking the intracellular cytosolic pH near neutrality (25,26)), (ii) pH 5.5 (mimicking the mildly acidic pH in immature and mature secretory granules (26,27)), and (iii) 0.01 N HCl (0.01 N HCl, 150 mM NaCl, pH 2.0). All samples contain 0.1% sodium azide.…”

Thea3 isoform of V-ATPase regulates insulin secretion from pancreatic β-cells

“…Because the conversion of proinsulin to insulin is strictly dependent on a low pH (Orci et al, 1994;Smeekens et al, 1992), we presumed that loss of the V-ATPase a3 isoform would result in abnormal luminal acidification, and thereby affect insulin processing and maturation. Insulin processing was examined using immunoblotting analysis of isolated islets.…”

“…In this study, we focused on the insulincontaining secretory granules in pancreatic ␤-cells. Previous studies suggested that the V-ATPase function might be important for the appropriate processing and maturation of insulin (Hutton, 1994;Orci et al, 1994;Orci et al, 1986;Smeekens et al, 1992). However, a mutant mouse with defective V-ATPase subunits in their secretory vesicles could produce the mature active insulin, whereas the mutant was less competent for hormone secretion following stimulation both in vivo and in vitro.…”

“…For instance, processing of insulin from proinsulin initiates in clathrin-coated secretory vesicles (Orci et al, 1987) by the activities of two enzymes, PC2 and PC3. Because PC3 shows a strict pH dependence (Orci et al, 1994;Smeekens et al, 1992), the acidification inside the secretory vesicles has fundamental importance for maintenance of whole-body homeostasis. It has been suggested for decades that the proton gradients might be involved in the fusion of secretory vesicles to the target membrane (Al-Awqati, 1986;Burgess and Kelly, 1987).…”