MFS transportome of the human pathogenic yeast Candida albicans

<p>Abstract</p> <p>Background</p> <p>The major facilitator superfamily (MFS) is one of the two largest superfamilies of membrane transporters present ubiquitously in bacteria, archaea, and eukarya and includes members that function as uniporters, symporters or antiporters. We report here the complete transportome of MFS proteins of a human pathogenic yeast <it>Candida albicans</it>.</p> <p>Results</p> <p>Computational analysis of <it>C. albicans </it>genome enabled us to identify 95 potential MFS proteins which clustered into 17 families using Saier's Transport Commission (TC) system. Among these SP, DHA1, DHA2 and ACS represented major families consisting of 22, 22, 9 and 16 members, respectively. Family designations in <it>C. albicans </it>were validated by subjecting <it>Saccharomyces cerevisiae </it>genome to TC system. Based on the published available genomics/proteomics data, 87 of the putative MFS genes of <it>C. albicans </it>were found to express either at mRNA or protein levels. We checked the expression of the remaining 8 genes by using RT-PCR and observed that they are not expressed under basal growth conditions implying that either these 8 genes are expressed under specific growth conditions or they may be candidates for pseudogenes.</p> <p>Conclusion</p> <p>The <it>in silico </it>characterisation of MFS transporters in <it>Candida albicans </it>genome revealed a large complement of MFS transporters with most of them showing expression. Considering the clinical relevance of <it>C. albicans </it>and role of MFS members in antifungal resistance and nutrient transport, this analysis would pave way for identifying their physiological relevance.</p

Purification and characterization of the N-terminal nucleotide binding domain of an ABC drug transporter of Candida albicans: uncommon cysteine 193 of Walker A is critical for ATP hydrolysis

The Candida drug resistance protein Cdr1p (~170 kDa) is a member of ATP binding cassette (ABC) superfamily of drug transporters, characterized by the presence of 2 nucleotide binding domains (NBD) and 12 transmembrane segments (TMS). NBDs of these transporters are the hub of ATP hydrolysis activity, and their sequence contains a conserved Walker A motif (GxxGxGK&#x0332;S/T). Mutations of the lysine residue within this motif abrogate the ability of NBDs to hydrolyze ATP. Interestingly, the sequence alignments of Cdr1p NBDs with other bacterial and eukaryotic transporters reveal that its N-terminal NBD contains an unusual Walker A sequence (GRPGAGC&#x0332;ST), as the invariant lysine is replaced by a cysteine. In an attempt to understand the significance of this uncommon positioning of cysteine within the Walker A motif, we for the first time have purified and characterized the N-terminal NBD (encompassing first N-terminal 512 amino acids) of Cdr1p as well as its C193A mutant protein. The purified NBD-512 protein could exist as an independent functional general ribonucleoside triphosphatase with strong divalent cation dependence. It exhibited ATPase activity with an apparent Km in the 0.8-1.0 mM range and Vmax in the range of 147-160 nmol min-1 (mg of protein)-1. NBD-512-associated ATPase activity was also sensitive to inhibitors such as vanadate, azide, and NEM. The Mut-NBD-512 protein (C193A) showed a severe impairment in its ability to hydrolyze ATP (95%); however, no significant effect on ATP (TNP-ATP) binding was observed. Our results show that C193 is critical for N-terminal NBD-mediated ATP hydrolysis and represents a unique feature distinguishing the ATP-dependent functionality of the ABC transporters of fungi from those found in bacteria and other eukaryotes