Plášek Jaromír, Babuka David, Gášková Dana, Jančíková Iva, Zahumenský Jakub, Hoefer Milan
Faculty of Mathematics and Physics, Charles University, Ke Karlovu 3, 12116, Prague, Czech Republic.
Institute of Cellular and Molecular Botany, University of Bonn, Kirschallee 1, 53115, Bonn, Germany.
J Bioenerg Biomembr. 2017 Jun;49(3):273-279. doi: 10.1007/s10863-017-9710-3. Epub 2017 Apr 12.
Yeast cells exhibit a negative surface potential due to negative charges at the cell membrane surface. Consequently, local concentrations of cations at the periplasmic membrane surface may be significantly increased compared to their bulk environment. However, in cell suspensions only bulk concentrations of cations can be measured directly. Here we present a novel method enabling the assessment of local pH at the periplasmic membrane surface which can be directly related to the underlying cell surface potential. In this proof of concept study using Saccharomyces cerevisiae cells with episomally expressed pH reporter, pHluorin, intracellular acidification induced by the addition of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) was measured using synchronously scanned fluorescence spectroscopy (SSF). The analysis of titration curves revealed that the pH at the periplasmic surface of S. cerevisiae cells was about two units lower than the pH of bulk medium. This pH difference was significantly decreased by increasing the ionic strength of the bulk medium. The cell surface potential was estimated to amount to -130 mV. Comparable results were obtained also with another protonophore, pentachlorophenol (PCP).
由于细胞膜表面带负电荷，酵母细胞呈现负表面电位。因此，与整体环境相比，周质膜表面阳离子的局部浓度可能会显著增加。然而，在细胞悬液中，只能直接测量阳离子的整体浓度。在此，我们提出一种新方法，能够评估周质膜表面的局部pH值，该值可直接与潜在的细胞表面电位相关。在这项概念验证研究中，使用携带附加表达的pH报告基因pHluorin的酿酒酵母细胞，通过同步扫描荧光光谱法（SSF）测量添加质子载体羰基氰化物间氯苯腙（CCCP）诱导的细胞内酸化。滴定曲线分析表明，酿酒酵母细胞周质表面的pH值比整体培养基的pH值低约两个单位。通过增加整体培养基的离子强度，这种pH差异显著降低。估计细胞表面电位为-130 mV。使用另一种质子载体五氯苯酚（PCP）也获得了类似结果。
