Engineering a Cysteine-Deficient Functional Candida albicans Cdr1 Molecule Reveals a Conserved Region at the Cytosolic Apex of ABCG Transporters Important for Correct Folding and Trafficking of Cdr1

Abstract

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. 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.

Novel ABC Transporter Associated with Fluconazole Resistance in Aging of Cryptococcus neoformans

“…Heterologous protein expression in yeast model systems, such as S. cerevisiae , has enabled functional analysis of specific proteins and has been previously employed to study efflux pumps in a variety of fungi [ 6 , 20 , 38 ]. Interestingly, Afr3 effectively exports both Rhodamine 6G and Nile Red out of the mutated S. cerevisiae strain, whereas the Afr1 pump only exports Nile Red and not Rhodamine 6G efflux [ 14 ].…”

“…Based on Afr3’s six transmembrane domains, we propose that Afr3 may be expressed in membranes of organelles in the cytoplasm. ABC transporters of Candida glabrata , Candida albicans , S. cerevisiae , and Aspergillus fumigatus [ 37 , 38 , 39 , 40 ] have been shown to localize in the membranes of mitochondria and the vacuole [ 41 , 42 ]. Future studies are necessary to analyze the subcellular localization of Afr3, especially during aging, since Afr3 expression negatively affects the replicative life span.…”

Functional Expression of Recombinant Candida auris Proteins in Saccharomyces cerevisiae Enables Azole Susceptibility Evaluation and Drug Discovery

“…The ATPase activity of recombinant strains overexpressing Cdr1 was measured in the presence and absence of OM, using crude membranes isolated from 40 mL YPD cultures, as previously described [ 47 , 48 , 49 , 50 ]. Crude membranes (0.1–0.2 µg of protein per well) were incubated with assay cocktail (50 mM Tris-HCl pH 7.5, 50 mM potassium nitrate, 0.2 mM ammonium molybdate, 5 mM sodium azide) and 6mM Mg-ATP in a total volume of 120 µL.…”

Clorgyline Analogs Synergize with Azoles against Drug Efflux in Candida auris

“…ATPase activity was determined as previously described [ 25 , 83 , 84 , 85 ]. Crude membranes (obtained by cell breakage and differential centrifugation) of recombinant strains were co-incubated with inhibitor in three-fold serial dilution in assay buffer.…”