Noninvasive measurement of the pH of the endoplasmic reticulum at rest and during calcium release

Abstract

The pH within individual organelles of the secretory pathway is believed to be an important determinant of their biosynthetic activity. However, little is known about the determinants and regulation of the pH in the secretory organelles, which cannot be readily accessed by [H ؉ ]-sensitive probes. We devised a procedure for the dynamic, noninvasive measurement of pH in the lumen of the endoplasmic reticulum in intact mammalian cells. A recombinant form of the B subunit of Shiga toxin, previously modified to include a carboxyl-terminal KDEL sequence and a pH-sensitive f luorophore, was used for a two-stage delivery strategy. Retrograde traffic of endogenous lipids was harnessed to target this protein to the Golgi complex, followed by retrieval to the endoplasmic reticulum (ER) by KDEL receptors. Immunof luorescence and immunoelectron microscopy were used to verify the subcellular localization of the modified B fragment. Fluorescence ratio imaging and two independent calibration 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 found to be highly permeable to H ؉ (equivalents), so that the prevailing [H ؉ ] is susceptible to alterations in the cytosolic pH. Plasmalemmal acid-base transporters were shown to indirectly regulate the endoplasmic reticulum pH.

Functional in Vitro Analysis of the ERO1 Protein and Protein-disulfide Isomerase Pathway

“…1, C and D). This suggested that PDI is fine-tuned to catalyze oxidative folding near the physiological pH in the ER (27). This result is consistent with previous data, which suggests the isomerase and oxidase activities of PDI are most efficient at pH 7 when using RNase or a small peptide as model substrates (18,19).…”

Nematotoxicity of Marasmius oreades Agglutinin (MOA) Depends on Glycolipid Binding and Cysteine Protease Activity

“…In accordance with such a hypothesis, the combination of neutral pH (37) and a Ca 2ϩ concentration of Ͼ100 M in the endoplasmic reticulum (38) meets the requirements of MOA to exhibit protease activity. On the other hand, MOA is not expected to be catalytically active at the neutral pH but low Ca 2ϩ concentration (ϳ100 nM) of the cytosol (37,39), which allows storage of this toxic protein in an inactive form in the cytoplasm of the fungal cell. Similarly, the slightly acidic pH in the intestinal lumen of the nematode should inhibit the proteolytic activity of MOA despite the presumably high Ca 2ϩ concentrations in this compartment (33).…”

Nox5 and the Regulation of Cellular Function

“…It is known that a reduction in pH can promote the formation of hydroperoxyl radicals which can then traverse biological membranes. However, the pH of the endoplasmic reticulum is near neutral and is effectively connected to pH changes in the cytoplasmic environment (41), and thus the vast majority of superoxide formed is likely to remain in the charged watersoluble and membrane-impermeant state. An alternative pathway has recently been revealed by studies showing that endosomal and plasma membrane chloride channels rapidly conduct superoxide from intracellular organelles to the extracellular space (31,52) (Fig.…”