Functional reconstitution of a purified proline permease from Candida albicans: interaction with the antifungal cispentacin.
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 microM. 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-proline binding and translocation in reconstituted proteoliposomes. However, the uptake of L-proline in proteoliposomes reconstituted with the purified protein displayed monophasic kinetics with an apparent Km of 40 microM.
Jethwaney
Deepa
D
School of Life Sciences, Jawaharlal Nehru University,New Delhi-110067,India.
H Fer
Milan
M
Botanisches Institut, Universit�t Bonn,Kirschallee 1, D-53115 Bonn,Germany.
Khaware
Raj K
RK
School of Life Sciences, Jawaharlal Nehru University,New Delhi-110067,India.
Prasad
Rajendra
R
School of Life Sciences, Jawaharlal Nehru University,New Delhi-110067,India.
D026921:Amino Acid Transport Systems, Neutral; D000935:Antifungal Agents / Q000494:pharmacology*; D001692:Biological Transport; D002176:Candida albicans / Q000201:enzymology*; D002846:Chromatography, Affinity; D003515:Cycloleucine / Q000031:analogs & derivatives / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D007700:Kinetics; D008564:Membrane Potentials; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D011392:Proline / Q000378:metabolism*; D011510:Proteolipids / Q000187:drug effects / Q000378:metabolism; D012995:Solubility; D014634:Valinomycin / Q000494:pharmacology
D026921:Amino Acid Transport Systems, Neutral; D000935:Antifungal Agents; D007476:Ionophores; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins; D011510:Proteolipids; C020485:proteoliposomes; D003515:Cycloleucine; D014634:Valinomycin; C065594:cispentacin; C033082:proline transporter; D011392:Proline; D005033:Ethylmaleimide