Measurement of cytosolic, mitochondrial, and Golgi pH in single living cells with green fluorescent proteins
Measurement of cytosolic, mitochondrial, and Golgi pH in single living cells with green fluorescent proteins
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DOI：
10.1073/pnas.95.12.6803
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发表时间：
1998-06-09
影响因子：
11.1
通讯作者：
Tsien, RY
中科院分区：
文献类型：
--
作者：
Llopis, J;McCaffery, JM;Tsien, RY
许多细胞事件依赖于H+离子在膜结合细胞器上的紧密分隔分布。然而，活细胞内细胞器pH的测量一直很少，Aequorea Victoria绿色荧光蛋白的几个突变体显示出依赖于pH的吸收和荧光发射，其表观pKa值从6.15(突变F64L/S65T/H231L)和6.4(K26R/F64L/S65T/Y66W/N146I/M153T/V163A/N164H/H231L)到显著的7.1(S65G/S72A/T203Y/H231L)。我们利用细胞色素C氧化酶IV亚基的靶向信号，通过与半乳糖基转移酶融合，将这些GFP靶向到胞浆+核、中间/反式高尔基体和线粒体基质，在培养的细胞中，这些cDNA在光镜和电子显微镜下显示了预期的亚细胞定位，并报告了用离子载体原位校准的局部pH。我们监测了HeLa细胞的胞浆和胞核pH，以及HeLa细胞和新生大鼠心肌细胞的线粒体基质pH。在稳态(在HeLa细胞中校准为6.58)和各种操作后测量内侧/跨高尔基体的pH值。这些结果表明，完整细胞的高尔基体膜对H+具有相对的通透性，而Cl-作为H+运输的反离子，可能有助于维持电子中和性，使用基因融合和转移技术将GFP工程到特定的亚细胞位置或组织靶点的顺应性应该使我们能够检测以前无法到达的位置的pH。
Many cellular events depend on a tightly compartmentalized distribution of H+ ions across membrane-bound organelles. However, measurements of organelle pH in living cells have been scarce, Several mutants of the Aequorea victoria green fluorescent protein (GFP) displayed a pH-dependent absorbance and fluorescent emission, with apparent pKa values ranging from 6.15 (mutations F64L/S65T/H231L) and 6.4 (K26R/F64L/S65T/Y66W/N146I/M153T/V163A/N164H/H231L) to a remarkable 7.1 (S65G/S72A/T203Y/H231L). We have targeted these GFPs to the cytosol plus nucleus, the medial/trans-Golgi by fusion with galactosyltransferase, and the mitochondrial matrix by using the targeting signal from subunit IV of cytochrome c oxidase, Cells in culture transfected with these cDNAs displayed the expected subcellular localization by light and electron microscopy and reported local pH that was calibrated in situ with ionophores. We monitored cytosolic and nuclear pH of HeLa cells, and mitochondrial matrix pH in HeLa cells and in rat neonatal cardiomyocytes. The pH of the medial/trans-Golgi was measured at steady-state (calibrated to be 6.58 in HeLa cells) and after various manipulations. These demonstrated that the Golgi membrane in intact cells is relatively permeable to H+, and that Cl- serves as a counter-ion for H+ transport and likely helps to maintain electroneutrality, The amenability to engineer GFPs to specific subcellular locations or tissue targets using gene fusion and transfer techniques should allow us to examine pH at sites previously inaccessible.