Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1999 Aug;181(15):4644-52.
doi: 10.1128/JB.181.15.4644-4652.1999.

The Saccharomyces cerevisiae weak-acid-inducible ABC transporter Pdr12 transports fluorescein and preservative anions from the cytosol by an energy-dependent mechanism

C D Holyoak  1 , D Bracey, P W Piper, K Kuchler, P J Coote
Affiliations

The Saccharomyces cerevisiae weak-acid-inducible ABC transporter Pdr12 transports fluorescein and preservative anions from the cytosol by an energy-dependent mechanism

C D Holyoak et al. J Bacteriol. 1999 Aug.

Abstract

Growth of Saccharomyces cerevisiae in the presence of the weak-acid preservative sorbic acid results in the induction of the ATP-binding cassette (ABC) transporter Pdr12 in the plasma membrane (P. Piper, Y. Mahe, S. Thompson, R. Pandjaitan, C. Holyoak, R. Egner, M. Muhlbauer, P. Coote, and K. Kuchler, EMBO J. 17:4257-4265, 1998). Pdr12 appears to mediate resistance to water-soluble, monocarboxylic acids with chain lengths of from C(1) to C(7). Exposure to acids with aliphatic chain lengths greater than C(7) resulted in no observable sensitivity of Deltapdr12 mutant cells compared to the parent. Parent and Deltapdr12 mutant cells were grown in the presence of sorbic acid and subsequently loaded with fluorescein. Upon addition of an energy source in the form of glucose, parent cells immediately effluxed fluorescein from the cytosol into the surrounding medium. In contrast, under the same conditions, cells of the Deltapdr12 mutant were unable to efflux any of the dye. When both parent and Deltapdr12 mutant cells were grown without sorbic acid and subsequently loaded with fluorescein, upon the addition of glucose no efflux of fluorescein was detected from either strain. Thus, we have shown that Pdr12 catalyzes the energy-dependent extrusion of fluorescein from the cytosol. Lineweaver-Burk analysis revealed that sorbic and benzoic acids competitively inhibited ATP-dependent fluorescein efflux. Thus, these data provide strong evidence that sorbate and benzoate anions compete with fluorescein for a putative monocarboxylate binding site on the Pdr12 transporter.

PubMed Disclaimer

Figures

FIG. 1
FIG. 1
Comparison of the growth inhibition of S. cerevisiae FY1679-28C, the isogenic parent (open bars), and YYM19, the Δpdr12 mutant (solid bars), upon exposure to a range of carboxylic acids with carbon chain lengths of C1 to C10. Growth was determined in a Labsystems Bioscreen apparatus as a detectable increase in optical density (600 nm) compared to the initial value. Arrows indicate that no growth was detected after 27 days of incubation at 30°C in YEPD (pH 4.5). A representative result of at least two replicate experiments is shown.
FIG. 2
FIG. 2
Efflux of fluorescein from S. cerevisiae FY1679-28c, the isogenic parent (■), and YYM19, the Δpdr12 mutant (□), resuspended in 50 mM HEPES-NaOH (pH 7.0), upon the addition of 10 mM glucose. Prior to loading of the cells with FDA, both FY1679-28c and YYM19 were grown in either YEPD (pH 4.5) with 0.45 mM sorbic acid to induce Pdr12 (A) or YEPD (pH 4.5) alone (B). The supernatant fluorescence intensity was collected at an excitation wavelength of 435 nm (a pH-independent point for fluorescein). Each datum point represents the mean and the standard deviation of three independent measurements.
FIG. 3
FIG. 3
Visualization of changes in the level of intracellular fluorescein after glucose addition in populations of S. cerevisiae FY1679-28c and YMM19 resuspended in 50 mM HEPES-NaOH (pH 7.0). Simultaneous phase-contrast and fluorescence images (excitation line, 488 nm) were obtained by CSLM. Images were taken prior to the addition of glucose (control) and at 0.5 and 2.0 h after the addition of 10 mM glucose to the cell suspensions. Both FY1679-28c and YYM19 were grown in YEPD (pH 4.5) in the presence of 0.45 mM sorbic acid prior to the loading with FDA. Representative images from a number of experiments are shown.
FIG. 4
FIG. 4
Glucose-induced (10 mM) efflux of fluorescein from S. cerevisiae FY1679-28c (solid symbols) and YYM19 (open symbols) resuspended in 50 mM HEPES-NaOH (pH 7.0) in the absence (●, ○) or presence (■, □) of 1 mM sodium orthovanadate (added 5 min prior to the glucose addition). Both FY1679-28c and YYM19 were grown in YEPD (pH 4.5) in the presence of 0.45 mM sorbic acid prior to the loading with FDA. Each datum point represents the mean and the standard deviation of three independent experiments.
FIG. 5
FIG. 5
Glucose-induced (10 mM) efflux of fluorescein from cells of S. cerevisiae FY1679-28c resuspended in 50 mM HEPES-NaOH (pH 5.5) in the presence of 0 mM (■), 0.9 mM (□), and 1.8 mM (○) sorbic acid (A) and 0 mM (■), 0.9 mM (□), and 1.8 mM (○) benzoic acid (B). Both sorbic acid and benzoic acid were added 5 min prior to the addition of glucose. FY1679-28c was grown in YEPD (pH 4.5) in the presence of 0.45 mM sorbic acid prior to the loading with FDA. Each datum point represents the mean and the standard deviation of three independent experiments.
FIG. 6
FIG. 6
Efflux of fluorescein from S. cerevisiae FY1679-28c resuspended in 50 mM HEPES-NaOH (pH 7.0) upon the addition of 10 mM glucose in the presence of 0 mM (■), 0.9 mM (□), and 1.8 mM (○) sorbic acid. Sorbic acid was added 5 min prior to the addition of glucose. FY1679-28c was grown in YEPD (pH 4.5) in the presence of 0.45 mM sorbic acid prior to the loading with FDA. Each datum point represents the mean and the standard deviation of three independent experiments.
FIG. 7
FIG. 7
Lineweaver-Burk plots illustrating competitive inhibition of glucose-induced (10 mM) efflux of fluorescein from S. cerevisiae FY1679-28c resuspended in 50 mM HEPES-NaOH (pH 5.5) by 0 mM (■), 0.9 mM (□), and 1.8 mM (○) sorbic acid (A) and 0 mM (■), 0.9 mM (□), and 1.8 mM (○) benzoic acid (B). Both sorbic acid and benzoic acid were added 5 min prior to the addition of glucose. FY1679-28c was grown in YEPD (pH 4.5) in the presence of 0.45 mM sorbic acid prior to the loading with FDA. Rates were calculated from the slope of the linear region of plots showing glucose-induced fluorescein efflux in the presence of increasing concentrations of preservatives. Rate data was then plotted and analyzed by linear regression (Microsoft Excel, version 5.0; Microsoft Corp.) to calculate Km values describing Pdr12-mediated efflux of fluorescein in the presence of preservatives. Representative results are shown.
FIG. 8
FIG. 8
The effect of exposure to 0.9 mM sorbic acid on the growth (solid symbols) and pHin (open symbols) of unadapted and preservative-adapted (pregrown in SD medium [pH 4.5] in the presence of 0.45 mM sorbic acid) cells of S. cerevisiae FY1679-28c growing in SD medium at pH 4.5 at 30°C. At the start of the experiment, the appropriate cells were inoculated into three separate flasks, with or without 0.9 mM sorbic acid, to give an identical starting optical density (600 nm) of 0.35. The growth (monitored by measuring the change in optical density at 600 nm) and pHin were measured in an untreated, control culture (▴, ▵), while unadapted cells were exposed to 0.9 mM sorbic acid (●, ○) and adapted cells were exposed to 0.9 mM sorbic acid (■, □). The actual value for pHin at the start of the experiment was approximately 6.0. Representative results of two independent experiments are shown.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Balzi E, Goffeau A. Genetics and biochemistry of yeast multidrug resistance. Biochim Biophys Acta. 1994;1187:152–162. - PubMed
    1. Bissinger P H, Kuchler K. Molecular cloning and expression of the Saccharomyces cerevisiae STS1 gene product. A yeast ABC transporter conferring mycotoxin resistance. J Biol Chem. 1994;269:4180–4186. - PubMed
    1. Bolhuis H, van Veen H W, Molenaar D, Poolman B, Driessen A J M, Konings W N. Multidrug resistance in Lactococcus lactis: evidence for ATP-dependent drug extrusion from the inner leaflet of the cytoplasmic membrane. EMBO J. 1996;15:4239–4245. - PMC - PubMed
    1. Bolhuis H, van Veen H W, Poolman B, Driessen A J M, Konings W N. Mechanisms of multidrug transporters. FEMS Microbiol Rev. 1997;21:55–84. - PubMed
    1. Breeuwer P, Drocourt J-L, Rombouts F M, Abee T. Energy-dependent, carrier-mediated extrusion of carboxyfluorescein from Saccharomyces cerevisiae allows rapid assessment of cell viability by flow cytometry. Appl Environ Microbiol. 1994;60:1467–1472. - PMC - PubMed

MeSH terms