Weak Acid Permeation in Synthetic Lipid Vesicles and Across the Yeast Plasma Membrane

Abstract

We present a fluorescence-based approach for determination of the permeability of small molecules across the membranes of lipid vesicles and living cells. With properly designed experiments, the method allows us to assess the membrane physical properties both in vitro and in vivo. We find that the permeability of weak acids increases in the order of benzoic > acetic > formic > lactic, both in synthetic lipid vesicles and the plasma membrane of Saccharomyces cerevisiae, but the permeability is much lower in yeast (one to two orders of magnitude). We observe a relation between the molecule permeability and the saturation of the lipid acyl chain (i.e., lipid packing) in the synthetic lipid vesicles. By analyzing wild-type yeast and a manifold knockout strain lacking all putative lactic acid transporters, we conclude that the yeast plasma membrane is impermeable to lactic acid on timescales up to ∼2.5 h.

Figure 1

Kinetic data obtained with the calcein self-quenching assay (upper panel) and the pyranine pH assay (lower panel) using liposomes composed of POPE:POPG:POPC at a 2:1:1 weight ratio. The color code is the same for both panels. To see this figure in color, go online.

Figure 2

Kinetic data obtained with the calcein self-quenching assay using liposomes composed of POPE:POPG:POPC at a 2:1:1 weight ratio. To see this figure in color, go online.

Figure 3

Schematic representation of the acid-base equilibria inside and outside the vesicles and the fluxes of weak acids and water across the membrane. P_AH and P_H2O refer to the weak acid and water permeability, respectively; A is surface area of the vesicle; V(t) is the volume of the vesicle.

Figure 4

Permeability coefficients (cm/s) (yellow circles) of water and the weak acids in liposomes prepared from the POPE:POPG:POPC lipid mixture (2:1:1 weight ratio). Blue circles: permeability (cm/s) measured by Walter et al. using egg phosphatidylcholine lipid membranes (47). To see this figure in color, go online.

Figure 5

Permeability coefficients P (cm/s) (green circles and yellow triangles) of lactic and formic acid in liposomes prepared from lipid mixtures differing in degrees of acyl chain saturation (d); d is 1, 0.84, 0.67, 0.5, 0.34, and 0.17. The error bars displays the experimental error of the measured permeability coefficients. p-Values are normalized to the permeability coefficients at d = 1; P_formic = 7.4 × 10⁻³ cm/s and P_lactic = 0.12 × 10⁻³ cm/s at d = 1. To see this figure in color, go online.

Figure 6

pH kinetics measured in vivo with pHluorin expressed in the S. cerevisiae strains RA380 (upper panel) and MG10 (lower panel). The color code is the same for both panels. The fast kinetics (continuous line) was resolved in the stopped-flow measurements and the slow kinetics (dots) with the fluorometer. The stopped-flow kinetics is plotted as the fluorescence intensity ratio r_390/470 instead of pH. To see this figure in color, go online.