Molecular cloning and functional characterisation of a glucose transporter, CaHGT1, of Candida albicans

We have cloned the first glucose transporter CaHGT1 (Candida albicanshigh-affinity glucose transporter) of a pathogenic yeast, Candida albicans. The DNA sequence (GenBank accession number Y16834) analysis revealed an ORF encoding a novel protein of 545 amino acids with a predicted molecular mass of 60.67 kDa. The putative protein with 12 transmembrane domains has 51% identity with Kluyveromyces lactis high-affinity glucose transporter, HGT1. The protein signatures which are conserved and distinctive of the sugar transporters belonging to the major facilitator superfamily (MFS) were also found in CaHgt1p. When heterologously expressed, the ORF functionally complemented a mutant strain of Saccharomyces cerevisiae RE700A which was deleted in seven hexose transporter genes and thus was unable to grow or transport glucose. The expression of CaHGT1 in C. albicans showed a transcript of 1.6 kb which was enhanced in response to the human steroid hormone progesterone. Interestingly, the transcript levels were also enhanced in the presence of drugs, e.g. cycloheximide, chloramphenicol and benomyl. The results suggest that CaHGT1, which encodes a MFS protein, could be linked to the drug resistance phenomenon in C. albicans

Tetraphenylphosphonium is an indicator of negative membrane potential in Candida albicans

The characteristics of the uptake of lipophilic cations tetraphenylphosphonium (TPP+) into Candida albicans have been investigated to establish whether TPP+ can be used as a membrane potential probe for this yeast. A membrane potential (Δψ, negative inside) across the plasma membrane of C. albicans was indicated by the intracellular accumulation of TPP+. The steady-state distribution of TPP+ was reached within 60 min and varied according to the expected changes of Δψ. Agents known to depolarize membrane potential caused a rapid and complete efflux of accumulated TPP+. The initial influx of TPP+ was linear over a wide range of TPP+ concentrations (2.5-600 µM), indicating a non mediated uptake. Thus, TPP+ is a suitable Δψ probe for this yeast

The Electrochemical H+ Gradient in the Yeast Rhodotorula glutinis

The electrochemical gradient of protons, Δµ~H+, was estimated in the obligatory aerobic yeast Rhodotorula glutinis in the pH0 range from 3 to 8.5. The membrane potential, ΔΨ, was measured by steady-state distribution of the hydrophobic ions, tetraphenylphosphonium (TPP+) for negative ΔΨ above pH0 4.5, and thiocyanate (SCN-) for positive ΔΨ below pH0 4.5. The chemical gradient of H+ was determined by measuring the chemical shift of intracellular Pi by 31P-NMR at given pH0 values. The values of pHi increased almost linearly from 7.3 at pH0 3 to 7.8 at pH0 8.5. In the physiological pH0 range from 3.5 to 6, Δµ~H+ was fairly constant at values between 17-18 KJ mol-1, gradually decreasing at pH0 above 6. In deenergized cells, the intracellular pHi decreased to values as low as 6, regardless of whether the cell suspension was buffered at pH0 4.5 or 7.5. There was no membrane potential detectable in deenergized cells.

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 µM. 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-ethylmaIeimide, 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 µM