ASSAY OF VACUOLAR PH IN YEAST AND IDENTIFICATION OF ACIDIFICATION-DEFECTIVE MUTANTS
ASSAY OF VACUOLAR PH IN YEAST AND IDENTIFICATION OF ACIDIFICATION-DEFECTIVE MUTANTS
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DOI：
10.1073/pnas.86.18.7027
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发表时间：
1989-09-01
影响因子：
11.1
通讯作者：
JONES, EW
中科院分区：
文献类型：
--
作者：
PRESTON, RA;MURPHY, RF;JONES, EW
作为酿酒酵母中液泡（溶酶体）的生物发生和功能的遗传分析的一部分，开发了液泡pH测定法，并用于鉴定液泡酸化缺陷突变体。用6-羧基荧光素与膜渗透前体6-羧基荧光素二乙酸酯标记胆甾醇。双激发流式细胞术用于校准体内6-羧基荧光素荧光的pH依赖性。在几种不同条件下生长的细胞中，野生型酵母中的胆甾醇是弱酸性的，pH 6.2。用6-羧基荧光素标记的培养物通过荧光比率显微镜进行筛选，以检测具有与血管酸化相关的缺陷的突变体。一个隐性的核突变，vph 1 -1，造成异常高vascuolar pH值为6.9，通过流式细胞仪测定，并消除了空泡吸收的弱碱性奎纳克林。酸化在pep 12：：LEU 2突变体出现缺陷的荧光比率显微镜和奎纳克林摄取测定，但在pep 12：：LEU 2突变体的血管pH值几乎是正常的（pH值6.3）在流式细胞仪检测。
As part of a genetic analysis of the biogenesis and function of the vacuole (lysosome) in the yeast Saccharomyces cerevisiae, assays of vacuolar pH were developed and used to identify mutants defective in vacuolar acidification. Vacuoles were labeled with 6-carboxyfluorescein with the membrane-permeant precursor 6-carboxyfluorescein diacetate. Dual-excitation flow cytometry was used to calibrate the pH-dependence of 6-carboxyfluorescein fluorescence in vivo. Vacuoles in wild-type yeast were mildly acidic, pH 6.2, in cells grown under several different conditions. Cultures labeled with 6-carboxyfluorescein were screened by fluorescence-ratio microscopy to detect mutants that had defects related to vascuolar acidification. A recessive nuclear mutation, vph1-1, caused an abnormally high vascuolar pH of 6.9, as assayed by flow cytometry, and eliminated vacuolar uptake of the weak base quinacrine. Acidification in a pep12::LEU2 mutant appeared defective by fluorescence-ratio microscopy and quinacrine-uptake assays, but the vascuolar pH in the pep12::LEU2 mutant was nearly normal (pH 6.3) in flow cytometric assays.