Abstract
ABSTRACTPleiotropic drug resistance (PDR) ATP-binding cassette (ABC) transporters of the ABCG family are eukaryotic membrane proteins that pump an array of compounds across organelle and cell membranes. Overexpression of the archetype fungal PDR transporter Cdr1 is a major cause of azole antifungal drug resistance in Candida albicans, a significant fungal pathogen that can cause life-threatening invasive infections in immunocompromised individuals. To date, no structure for any PDR transporter has been solved. The objective of this project was to investigate the role of the 23 Cdr1 cysteine residues in the stability, trafficking, and function of the protein when expressed in the eukaryotic model organism, Saccharomyces cerevisiae. The biochemical characterization of 18 partially cysteine-deficient Cdr1 variants revealed that the six conserved extracellular cysteines were critical for proper expression, localization, and function of Cdr1. They are predicted to form three covalent disulfide bonds that stabilize the large extracellular domains of fungal PDR transporters. Our investigations also revealed a novel nucleotide-binding domain motif, GX2[3]CPX3NPAD/E, at the peripheral cytosolic apex of ABCG transporters that possibly contributes to the unique ABCG transport cycle. With this knowledge, we engineered an “almost cysteine-less,” yet fully functional, Cdr1 variant, Cdr1P-CID, that had all but the six extracellular cysteines replaced with serine, alanine, or isoleucine (C1106I of the new motif). It is now possible to perform cysteine-cross-linking studies that will enable more detailed biochemical investigations of fungal PDR transporters and confirm any future structure(s) solved for this important protein family.IMPORTANCE Overexpression of the fungal pleiotropic drug resistance (PDR) transporter Cdr1 is a major cause of antifungal drug resistance in Candida albicans, a significant fungal pathogen that can cause life-threatening invasive infections in immunocompromised individuals. To date, no structure for any PDR ABC transporter has been solved. Cdr1 contains 23 cysteines; 10 are cytosolic and 13 are predicted to be in the transmembrane or the extracellular domains. The objective of this project was to create, and biochemically characterize, CDR1 mutants to reveal which cysteines are most important for Cdr1 stability, trafficking, and function. During this process we discovered a novel motif at the cytosolic apex of PDR transporters that ensures the structural and functional integrity of the ABCG transporter family. The creation of a functional Cys-deficient Cdr1 molecule opens new avenues for cysteine-cross-linking studies that will facilitate the detailed characterization of an important ABCG transporter family member.
Highlights
Pleiotropic drug resistance (PDR) ATP-binding cassette (ABC) transporters of the ABCG family are eukaryotic membrane proteins that pump an array of compounds across organelle and cell membranes
This study describes the creation of a functional, “almost” Cys-less, Cdr1 molecule and the discovery of a novel motif that is conserved among all ABCG transporters and provides a pivotal contact point between the two nucleotide binding domain (NBD) at their cytosolic apex
In order to determine the effect of replacing cysteine residues with either alanine or serine on Cdr1 expression and localization in S. cerevisiae, a green fluorescent protein (GFP) tag was fused to the C terminus of the CDR1 open reading frame (ORF)
Summary
Pleiotropic drug resistance (PDR) ATP-binding cassette (ABC) transporters of the ABCG family are eukaryotic membrane proteins that pump an array of compounds across organelle and cell membranes. Cdr contains 23 cysteines; 10 are cytosolic and 13 are predicted to be in the transmembrane or the extracellular domains The objective of this project was to create, and biochemically characterize, CDR1 mutants to reveal which cysteines are most important for Cdr stability, trafficking, and function. During this process we discovered a novel motif at the cytosolic apex of PDR transporters that ensures the structural and functional integrity of the ABCG transporter family. C. albicans Cdr has 1,501 amino acids, a molecular weight of 170 kDa, and an inverted nucleotide binding domain (NBD)-transmembrane domain (TMD) topology with a 2-fold pseudosymmetry [NBD-TMD]2 typical for full-size fungal PDR transporters (Fig. 1) These transporters have six extracellular loops (ELs) and four intracellular loops (ILs) connecting 12 individual transmembrane spans (TMSs). Many aspects of Cdr biology remain obscure [9, 29]
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