Active Trans-Plasma Membrane Water Cycling in Yeast Is Revealed by NMR
Active Trans-Plasma Membrane Water Cycling in Yeast Is Revealed by NMR
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DOI：
10.1016/j.bpj.2011.10.035
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发表时间：
2011-12-07
影响因子：
3.4
通讯作者：
Balschi, James A.
中科院分区：
文献类型：
--
作者：
Zhang, Yajie;Poirier-Quinot, Marie;Balschi, James A.
质膜水转运是一种重要的细胞现象。响应渗透梯度的净水运动改变细胞体积。水分子的稳态交换，没有净流量或体积变化，通过被动扩散通过磷脂双层和通过膜蛋白发生。这一假设进行了测试，质膜水交换也与ATP驱动的膜运输活动在酵母（酿酒酵母）。纵向（H2O）-H-1 NMR弛豫时间常数（T-1）值测定酵母悬浮液含有细胞外弛豫试剂。双位点交换分析将可逆交换动力学定量为平均细胞内水寿命（tau（i）），其中tau（-1）（i）是水流出的伪一级速率常数。为了调节细胞ATP，用95%0 -2/5%C02（0 -2）或95%N-2/5%C02（N-2）鼓泡酵母悬浮液。O-2期间ATP较高，25 ℃时tau（-1）（i）为3.1 s（-1）。转换为N-2后，ATP减少，tau（-1）（i）为1.8 s（-1）。主要的活性酵母离子转运蛋白是质膜H+-ATP酶。使用H+-ATP酶抑制剂ebselen或具有减少的H+-ATP酶的酵母遗传菌株的研究发现减少的tau（-1）（i），即不稳定的高ATP。稳态水交换与H+-ATP酶活性相关。在体积稳态下，水响应于代谢转运活性而循环穿过质膜。
Plasma membrane water transport is a crucial cellular phenomenon. Net water movement in response to an osmotic gradient changes cell volume. Steady-state exchange of water molecules, with no net flux or volume change, occurs by passive diffusion through the phospholipid bilayer and passage through membrane proteins. The hypothesis is tested that plasma membrane water exchange also correlates with ATP-driven membrane transport activity in yeast (Saccharomyces cerevisiae). Longitudinal (H2O)-H-1 NMR relaxation time constant (T-1) values were measured in yeast suspensions containing extracellular relaxation reagent. Two-site-exchange analysis quantified the reversible exchange kinetics as the mean intracellular water lifetime (tau(i)), where tau(-1)(i) is the pseudo-first-order rate constant for water efflux. To modulate cellular ATP, yeast suspensions were bubbled with 95%O-2/5%CO2 (O-2) or 95%N-2/5%CO2 (N-2). ATP was high during O-2, and tau(-1)(i) was 3.1 s(-1) at 25 degrees C. After changing to N-2, ATP decreased and tau(-1)(i) was 1.8 s(-1). The principal active yeast ion transport protein is the plasma membrane H+-ATPase. Studies using the H+-ATPase inhibitor ebselen or a yeast genetic strain with reduced H+-ATPase found reduced tau(-1)(i), notwith-standing high ATP. Steady-state water exchange correlates with H+-ATPase activity. At volume steady state, water is cycling across the plasma membrane in response to metabolic transport activity.