Functional reconstitution of a purified proline permease from Candida albicans: interaction with the antifungal cispentacin

Abstract

We have purified proline permease to homogeneity from Candida albicans using an L-proline-linked agarose matrix as an affinity column. The eluted protein produced two bands of 64 and 67 kDa by SDS-PAGE, whereas it produced a single band of 67 kDa by native PAGE and Western blotting. The apparent Km for L-proline binding to the purified protein was 153 pM. The purified permease was reconstituted into proteoliposomes and its functionality was tested by imposing a valinomycin-induced membrane potential. The main features of L-proline transport in reconstituted systems, viz. specificity and sensitivity to N-ethylmaleimide, were very similar to those of intact cells. The antifungal cispentacin, which enters C. albicans cells via an inducible proline permease, competitively inhibited the L-praline binding and translocation in reconstituted proteoliposomes. However, the uptake of Lproline in proteoliposomes reconstituted with the purified protein displayed monophasic kinetics with an apparent Km of 40 pM.

Induction of oxidative stress as a possible mechanism of the antifungal action of three phenylpropanoids

“…Cell‐free extracts of treated and control C. albicans ATCC 90028 were prepared according to the method described by Jethwaney et al (1997), with minor modifications. Cells (1 g wet weight) grown in YPD medium to the mid‐exponential phase in the presence of 0, 10, 50 and 100 μg mL −1 of test phenylpropanoids, were suspended in 2 mL grinding medium (250 mM sucrose, 10 mM Tris HCl, pH 7.5, 1 mM PMSF) and 2‐g glass beads (0.45 mm).…”

Deletion of transmembrane domain 12 ofCDR1, a multidrug transporter fromCandida albicans, leads to altered drug specificity: Expression of a yeast multidrug transporter in baculovirus expression system

“…Yeast plasma membranes were purified as described elsewhere (Jethwaney et al, 1997;Hubbard et al, 1986). Yeast cells were grown in YNB supplemented with required nutritional markers.…”

“…The pellet was resuspended in 10 m-Tris-Cl, pH 7·5 (crude membrane, CM). The CM fraction was further purified on a Percoll (Sigma, USA) gradient as described elsewhere (Jethwaney et al, 1997). The plasma membrane (PM) band was carefully aspirated off and washed twice in 10 m-Tris-Cl pH 7·5.…”

Asymmetric distribution of phosphatidylethanolamine inC. albicans : possible mediation byCDR1, a multidrug transporter belonging to ATP binding cassette (ABC) superfamily

“…Candida albicans ATCC 10261 was grown in YEPD medium (2% bactopeptone, 1% bactoyeast extract and 2% glucose) (Jethwaney et al, 1997). C. albicans strains CAF2-1 ( ura3::imm434/ URA3), DSY449 ( cdr1::hisG/ cdr1::hisG), DSY1025 ( cdr1::hisG/ cdr1::hisG, cdr2::hisG/ cdr2::hisG) were also maintained and grown in YEPD medium, as described earlier (Sanglard et al, 1997).…”