Kim J H, Johannes L, Goud B, Antony C, Lingwood C A, Daneman R, Grinstein S
Division of Gastroenterology and Nutrition, Research Institute, Hospital for Sick Children, 555 University Avenue, Toronto, ON, Canada M5G 1X8.
Proc Natl Acad Sci U S A. 1998 Mar 17;95(6):2997-3002. doi: 10.1073/pnas.95.6.2997.
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.
分泌途径中各个细胞器内的pH值被认为是其生物合成活性的重要决定因素。然而，对于分泌细胞器中pH值的决定因素和调节机制却知之甚少，因为[H⁺]敏感探针无法轻易进入这些细胞器。我们设计了一种方法，用于动态、非侵入性地测量完整哺乳动物细胞内质网腔中的pH值。使用了一种重组形式的志贺毒素B亚基，该亚基先前经过修饰，包含一个羧基末端KDEL序列和一个pH敏感荧光团，采用两阶段递送策略。利用内源性脂质的逆行运输将该蛋白靶向高尔基体复合体，随后通过KDEL受体将其回收至内质网（ER）。通过免疫荧光和免疫电子显微镜验证修饰后的B片段的亚细胞定位。应用荧光比率成像和两种独立的校准程序来原位测定内质网的pH值。我们发现内质网的pH值接近中性，并且在激动剂诱导的钙释放过程中不受影响。发现内质网对H⁺（当量）具有高度渗透性，因此主要的[H⁺]易受细胞质pH值变化的影响。已证明质膜酸碱转运蛋白间接调节内质网pH值。
