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 e luted 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 blotti ng. The apparent K-m for L-proline binding to the purified protein was 153 mu M. The purified permease was reconstituted into proteoliposome s and its functionality was tested by imposing a valinomycin-induced m embrane potential. The main features of L-proline transport in reconst ituted 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, co mpetitively inhibited the L-proline binding and translocation in recon stituted proteoliposomes. However, the uptake of L-proline in proteoli posomes reconstituted with the purified protein displayed monophasic k inetics with an apparent K-m of 40 mu M.