In situ measurements of the pH of mammalian peroxisomes using the fluorescent protein pHluorin
In situ measurements of the pH of mammalian peroxisomes using the fluorescent protein pHluorin
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DOI：
10.1074/jbc.m109003200
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发表时间：
2001-12-28
影响因子：
4.8
通讯作者：
Grinstein, S
中科院分区：
文献类型：
--
作者：
Jankowski, A;Kim, JH;Grinstein, S
过氧化物酶体是代谢活跃的细胞器，参与长链脂肪酸的氧化以及胆汁酸、胆固醇和醚磷脂的生物合成。尽管维持稳定的酸碱环境对于过氧化物酶体的正常功能至关重要，但过氧化物酶体 pH (pHP) 的测定仍然没有定论，并且对其调节知之甚少。为了原位测量完整过氧化物酶体的 pH 值，我们使用过氧化物酶体特异性羧基末端靶向序列 SKL，将绿色荧光蛋白的 pH 敏感突变体 (pHluorin-SKL) 选择性地递送到过氧化物酶体中。通过与过氧化物酶体标记物过氧化氢酶共定位来验证正确的靶向性。通过成像荧光显微镜观察过氧化物酶体，并将比率测量与使用离子载体或零点法的校准相结合来估计 pH(p)。 pH(p) 介于 6.9 和 7.1 之间，类似于细胞质 pH。对完整细胞中的胞质 pH 值或用链球菌溶血素 O 透化质膜后的胞质 pH 值进行操作，发现 pHP 发生平行变化，表明过氧化物酶体膜对 H+（等价物）具有高度渗透性。我们得出的结论是，过氧化物酶体不能独立调节其 pH 值，而是其较大的 H+ 渗透性有效地将它们与细胞质的缓冲库以及控制细胞质 pH 值的稳态机制连接起来。
Peroxisomes are metabolically active organelles that participate in the oxidation of long-chain fatty acids and in the biosynthesis of bile acids, cholesterol, and ether phospholipids. Even though maintenance of a stable acid-base milieu is essential for proper peroxisomal function, the determination of the peroxisomal pH (pHP) remains inconclusive, and little is known about its regulation. To measure the pH of intact peroxisomes in situ, we used the peroxisome-specific carboxyl-terminal targeting sequence, SKL, to deliver a pH-sensitive mutant of the green fluorescent protein (pHluorin-SKL) selectively into peroxisomes. Proper targeting was verified by colocalization with the peroxisomal marker catalase. Peroxisomes were visualized by imaging fluorescence microscopy, and ratiometric measurements were combined with calibration using ionophores or a null-point method to estimate pH(p). The pH(p) was between 6.9 and 7.1, resembling the cytosolic pH. Manipulation of the cytosolic pH in intact cells or after permeabilization of the plasmalemma with streptolysin O revealed that pHP changed in parallel, suggesting that the peroxisomal membrane is highly permeable to H+ (equivalents). We conclude that peroxisomes do not regulate their pH independently, but instead their large H+ permeability effectively connects them with the buffer reservoir of the cytoplasm and with the homeostatic mechanisms that control cytosolic pH.