期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1998
卷号：95
期号：6
页码：2997-3002
DOI：10.1073/pnas.95.6.2997
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：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 fluorophore, 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. Immunofluorescence 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.
