Plasma-membrane permeability of S. cerevisiae by PGSE NMR

Pavel Srb¹, Hana Elicharová², Larisa Starovoytova³, Jaroslav Kříž³,

Hana Sychrová² and Jan Lang¹

1Department of Low Temperature Physics, Faculty of Mathematics and Physics, Charles University, V Holešovičkách 2, 180 00 Prague 8, Czech Republic

2Department of Membrane Transport, Institute of Physiology AS CR, v.v.i., Videnska 1083,

CZ-14220, Prague 4, Czech Republic

3Institute of Macromolecular Chemistry AS CR, v.v.i., Heyrovského nám. 2

CZ-162 06, Praha 6, Czech Republic
pavel.srb@matfyz.cz

The plasma membrane is an essential component of all living cells. It is involved in cell integrity and volume maintenance, metabolism, signal regulation and transport. It is fully permeable for small uncharged molecules like water.

The principle of monitoring of the plasma-membrane fluidity relies on the distinction between extra- and intracellular water – which in fact requires some “labeling” of the molecules. The widely used and well established NMR method for measurement of exchange over plasma membrane utilizes doping of the extracellular compartment by paramagnetic agents which enhance relaxation of extracellular molecules [1, 2]. Alternatively, the difference in apparent translational diffusion coefficients can be used as a “labeling” method to distinguish extra- and intracellular water NMR signal. Such method was proposed and applied to monitor the temperature dependent plasma-membrane fluidity of bakers yeast [3]. The Filter Exchange spectroscopy (FEXSY) experiment comprises two building blocks formed by pulsed gradients of magnetic field. The amplitude and spacing of gradient pulses in first block are set to a fixed value to effectively suppress the NMR signal originating from extracellular water molecules. After short delay (10 – 400 ms) called mixing time, during which the water molecules exchange over plasma membrane, the second block is applied. Here the amplitude of gradient pulses is gradually incremented in sixteen steps in order to perform a standard NMR experiment for measurement of translational diffusion. The information about plasma-membrane fluidity is encoded in the water NMR signal intensity recorded for four values of mixing time. The most interesting parameter – the intracellular water life-time – is then obtained by fitting the theoretical equations (Eq. 2-12 in [3]) to experimental signal intensities.

We studied three S. cerevisiae deletion mutant strains (erg2, erg4 and erg6) which lack genes involved in the synthesis of ergosterol, the most important steroid compound present in the plasma membrane. Blocking of different steps of ergosterol synthesis leads to accumulation of various intermediates within the cell. Therefore the composition of plasma membrane differs among selected strains. We found the intracellular water life-time to be very similar for wild type S. cerevisiae and for erg2 mutant strain, while erg6 and erg4 mutants show significantly faster water exchange over plasma membrane.

[1] T.Conlon, R. Outhred. Biochim. Biophys. Acta., 288, 354-361, (1972)

[2] G. Benga, Prog. Biophys. Mol.Biol., 51, 193-245, (1988)

[3] I. Ǻslund, et al. J. Magn. Reson., 200, 291-295, (2009)