Comparative Roles of the Cell Wall and Cell Membrane in Limiting Uptake of Xenobiotic Molecules by Saccharomyces cerevisiae

Abstract

Using reversible electropermeabilization of cells and spheroplasts, we show that the cell wall and plasma membrane partly account for bleomycin resistance by acting as two independent barriers. We also report on the presence of a membrane protein that may be responsible for bleomycin internalization and toxicity in Saccharomyces cerevisiae.The antitumor drug bleomycin (BLM) was used to analyze the contributions of the cell wall and the cell membrane in limiting the uptake of hydrophilic cytotoxic molecules during the stationary phase of the yeast Saccharomyces cerevisiae. Two yeast strains with different sensitivities to BLM were tested: YPH-1 (MATa ura3-52 lys2-801am ade2-101oc) (14) and FY67 (MATa trp1-⌬36) (15).Yeast cells were grown to a density of approximately 10 8 cells/ml through shaking and aeration in complete YPD (yeast extract-peptane-dextrose) medium (13) at 30°C. We determined the percentage of reversibly permeabilized cells by the lucifer yellow test (6) and prepared spheroplasts as described previously (2) with lyticase from Sigma. A total of 5 ϫ 10 7 cells were electropermeabilized as described previously (1) in the absence of dithiothreitol by using eight 100-s square-wave pulses at a frequency of 1 Hz. Spheroplasts were electroporated in 1.2 M sorbitol-1 mM MgCl 2 -10 mM Tris-HCl (pH 7.5). No effect on survival was detected when electric fields from 0 to 2,000 V/cm were applied to either the cells or the spheroplasts. Optimal conditions were chosen such that the electric field resulted in 70 to 75% survival and electropermeabilization of approximately 85% of those surviving cells (i.e., 4,640 and 3,920 V/cm for FY67 and YPH-1 cells, respectively, and 4,300 and 3,900 V/cm for FY67 and YPH-1 spheroplasts, respectively) (Fig. 1). Our results show that the optimal permeabilization value is a function of the difference between the mean cell diameter for the two strains, as determined by flow cytometry (50.5 Ϯ 2.12 and 58 Ϯ 2.83, in arbitrary units, for FY67 and YPH-1 cells, respectively, and 49 Ϯ 2.15 and 56.5 Ϯ 1.41 for FY67 and YPH-1 spheroplasts, respectively). Our data also show that the removal of the cell wall does not significantly alter the cell diameter and has almost no influence on the optimal electrical field strength applied to the spheroplasts. Thus, the presence of the cell wall does not affect the electropermeabilization of the plasma membrane.Both yeast strains were exposed to BLM for 5 min at concentrations ranging from 0.3 nM to 100 M, with and without application of the electric pulses. In this study, a BLM-Fe complex in a 1:3 molar ratio was used to induce cytotoxicity in the yeasts (7). The cytotoxicity of BLM was determined by determination of the relative cloning efficiencies of the treated cells. The BLM concentration that induced 50% lethality (the minimal bactericidal concentration for 50% of the cells [MBC 50 ]) for the nonelectropermeabilized YPH-1 yeast cells was 4 M. The FY67 yeast cells were approximately 30 times more resistant to BLM, with an MBC 50 of 120 M ( Fig...

Newly Discovered and Characterized Antivirulence Compounds Inhibit Bacterial Mono-ADP-Ribosyltransferase Toxins

“…This was most obvious in the structure/activity relationships of NAP and its more polar derivative, 4-amino-NAP, and between PJ97A and the more polar PJ34, where the nonpolar parent compound was active against ExoA c in yeast, but the more polar derivative was considerably less active (Table 1; Fig. 1; see Tables S1 and S2) (1,28). None of the polar P-series compounds (P1 to P8) showed inhibitor efficacy in yeast against ExoA c , and in the V series only compound V23 showed modest protection in yeast, with weak protection afforded by compounds V15, V29, and V31.…”

“…None of the polar P-series compounds (P1 to P8) showed inhibitor efficacy in yeast against ExoA c , and in the V series only compound V23 showed modest protection in yeast, with weak protection afforded by compounds V15, V29, and V31. Yeasts grow on less complex media; are genetically simpler, lacking many redundant systems of mammalian cells; and provide more rapid feedback as a screening tool; however, yeasts are limited for identifying polar/ionizable inhibitors because their cell wall presents a barrier to the permeation of these compounds (1,28).…”

Electroporation in Biological Cell and Tissue: An Overview

“…Small molecules can freely diffuse through the cell wall; therefore, their loading into cytoplasm was not affected significantly by the presence of the cell wall in bacteria, yeasts, or plants. From those results, it was concluded that electroporation of cell membrane on itself is not affected by the presence of the cell wall (Ganeva et al 1995;Aouida et al 2003;Sauders et al 1995).…”

Electroporation of Cell Membranes: The Fundamental Effects of Pulsed Electric Fields in Food Processing

“…), which have to be used to determine cell electroporation. As examples of such molecules, sucrose (M w = 342.3 Da) [53,64], ethidium bromide (M w = 394.3 Da) [70], lucifer yellow (M w = 570 Da) [71], propidium iodide (M w = 668.4 Da) [47,63] or bleomycin (M w = 1,410-1,500 Da) [72], can be mentioned.…”