Active Trans-Plasma Membrane Water Cycling in Yeast Is Revealed by NMR

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.