NONINVASIVE MEASUREMENT OF THE PH OF THE ENDOPLASMIC-RETICULUM AT REST AND DURING CALCIUM-RELEASE

The pH within individual organelles of the secretory pathway is believ ed to be an important determinant of their biosynthetic activity. Howe ver, little is known about the determinants and regulation of the pH i n the secretory organelles, which cannot be readily accessed by [H+]-s ensitive probes. We devised a procedure for the dynamic, noninvasive m easurement of pH in the lumen of the endoplasmic reticulum in intact m ammalian cells. A recombinant form of the B subunit of Shiga toxin, pr eviously modified to include a carboxyl-terminal KDEL sequence and a p H-sensitive fluorophore, was used for a two-stage delivery strategy. R etrograde traffic of endogenous lipids was harnessed to target this pr otein to the Golgi complex, followed by retrieval to the endoplasmic r eticulum (ER) by KDEL receptors. Immunofluorescence and immunoelectron microscopy were used to verify the subcellular localization of the mo dified B fragment. Fluorescence ratio imaging and two independent cali bration procedures were applied to determine the pH of the ER in situ. We found that the pH of the endoplasmic reticulum is near neutral and is unaffected during agonist-induced release of calcium. The ER was f ound to be highly permeable to H+ (equivalents), so that the prevailin g [H+] is susceptible to alterations in the cytosolic pH. Plasmalemmal acid-base transporters were shown to indirectly regulate the endoplas mic reticulum pH.