Strayle J, Pozzan T, Rudolph H K
Institut für Biochemie der Universität Stuttgart, Pfaffenwaldring 55, D-70569 Stuttgart, Germany.
EMBO J. 1999 Sep 1;18(17):4733-43. doi: 10.1093/emboj/18.17.4733.
Over recent decades, diverse intracellular organelles have been recognized as key determinants of Ca(2+) signaling in eukaryotes. In yeast however, information on intra-organellar Ca(2+) concentrations is scarce, despite the demonstrated importance of Ca(2+) signals for this microorganism. Here, we directly monitored free Ca(2+) in the lumen of the endoplasmic reticulum (ER) of yeast cells, using a specifically targeted version of the Ca(2+)-sensitive photoprotein aequorin. Ca(2+) uptake into the yeast ER displayed characteristics distinctly different from the mammalian ER. At steady-state, the free Ca(2+) concentration in the ER lumen was limited to approximately 10 microM, and ER Ca(2+) sequestration was insensitive to thapsigargin, an inhibitor specific for mammalian ER Ca(2+) pumps. In pmr1 null mutants, free Ca(2+) in the ER was reduced by 50%. Our findings identify the secretory pathway pump Pmr1, predominantly localized in the Golgi, as a major component of ER Ca(2+) uptake activity in yeast.
在最近几十年里，多种细胞内细胞器已被确认为真核生物中钙信号传导的关键决定因素。然而，在酵母中，尽管钙信号对这种微生物具有重要意义，但关于细胞器内钙浓度的信息却很少。在这里，我们使用一种经过特殊靶向的钙敏感光蛋白水母发光蛋白，直接监测酵母细胞内质网（ER）腔内的游离钙。酵母内质网对钙的摄取表现出与哺乳动物内质网明显不同的特征。在稳态下，内质网腔内的游离钙浓度限制在约10微摩尔，并且内质网对钙的螯合作用对毒胡萝卜素不敏感，毒胡萝卜素是一种对哺乳动物内质网钙泵具有特异性的抑制剂。在pmr1基因缺失的突变体中，内质网中的游离钙减少了50%。我们的研究结果确定了主要定位于高尔基体的分泌途径泵Pmr1是酵母内质网钙摄取活性的主要组成部分。
