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ATP-binding cassette (ABC) transporters couple ATP binding and hydrolysis to the translocation of allocrites across membranes. Two shared nucleotide-binding sites (NBS) participate in this cycle. In asymmetric ABC pumps, only one of them hydrolyzes ATP, and the functional role of the other remains unclear. Using a drug-based selection strategy on the transport-deficient mutant L529A in the transmembrane domain of the Candida albicans pump Cdr1p; we identified a spontaneous secondary mutation restoring drug-translocation. The compensatory mutation Q1005H was mapped 60 Å away, precisely in the ABC signature sequence of the non-hydrolytic NBS. The same was observed in the homolog Cdr2p. Both the mutant and suppressor proteins remained ATPase active, but remarkably, the single Q1005H mutant displayed a two-fold reduced ATPase activity and a two-fold increased drug-resistance as compared to the wild-type protein, pointing at a direct control of the non-hydrolytic NBS in substrate-translocation through ATP binding in asymmetric ABC pumps., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Among the several mechanisms that contribute to MDR (multidrug resistance), the overexpression of drug-efflux pumps belonging to the ABC (ATP-binding cassette) superfamily is the most frequent cause of resistance to antifungal agents. The multidrug transporter proteins Cdr1p and Cdr2p of the ABCG subfamily are major players in the development of MDR in Candida albicans Because several genes coding for ABC proteins exist in the genome of C. albicans, but only Cdr1p and Cdr2p have established roles in MDR, it is implicit that the other members of the ABC family also have alternative physiological roles. The present study focuses on an ABC transporter of C. albicans, Mlt1p, which is localized in the vacuolar membrane and specifically transports PC (phosphatidylcholine) into the vacuolar lumen. Transcriptional profiling of the mlt1∆/∆ mutant revealed a down-regulation of the genes involved in endocytosis, oxidoreductase activity, virulence and hyphal development. High-throughput MS-based lipidome analysis revealed that the Mlt1p levels affect lipid homoeostasis and thus lead to a plethora of physiological perturbations. These include a delay in endocytosis, inefficient sequestering of reactive oxygen species (ROS), defects in hyphal development and attenuated virulence. The present study is an emerging example where new and unconventional roles of an ABC transporter are being identified., (© 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.)

The ABC transporter Cdr1 protein of Candida albicans, which plays a major role in antifungal resistance, has two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs). The 12 transmembrane helices of TMDs that are interconnected by extracellular and intracellular loops (ICLs) mainly harbor substrate recognition sites where drugs bind while cytoplasmic NBDs hydrolyze ATP which powers drug efflux. The coupling of ATP hydrolysis to drug transport requires proper communication between NBDs and TMDs typically accomplished by ICLs. This study examines the role of cytoplasmic ICLs of Cdr1p by rationally predicting the critical residues on the basis of their interatomic distances. Among nine pairs that fall within a proximity of <4 Å, an ion pair between K577 of ICL1 and E315 of NBD1 was found to be critical. The substitution, swapping and changing of the length or charge of K577 or E315 by directed mutagenesis led to a misfolded, non-rescuable protein entrapped in intracellular structures. Furthermore, the equipositional ionic pair-forming residues from ICL3 and NBD2 (R1260 and E1014) did not impact protein trafficking. These results point to a new role for ICL/NBD interacting residues in PDR ABC transporters in protein folding and trafficking., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

ATP-binding cassette (ABC) transporters help export various substrates across the cell membrane and significantly contribute to drug resistance. However, a recent study reported an unusual case in which the loss of anABCtransporter in Candida albicans, orf19.4531 (previously named ROA1), increases resistance against antifungal azoles, which was attributed to an altered membrane potential in the mutant strain. To obtain further mechanistic insights into this phenomenon, here we confirmed that the plasma membrane-localized transporter (renamed CDR6/ROA1 for consistency with C. albicans nomenclature) could efflux xenobiotics such as berberine, rhodamine 123, and paraquat. Moreover, a CDR6/ROA1 null mutant, NKKY101, displayed increased susceptibility to these xenobiotics. Interestingly, fluorescence recovery after photobleaching (FRAP) results indicated that NKKY101 mutant cells exhibited increased plasma membrane rigidity, resulting in reduced azole accumulation and contributing to azole resistance. Transcriptional profiling revealed that ribosome biogenesis genes were significantly up-regulated in the NKKY101 mutant. As ribosome biogenesis is a well-known downstream phenomenon of target of rapamycin (TOR1) signaling, we suspected a link between ribosome biogenesis and TOR1 signaling in NKKY101. Therefore, we grew NKKY101 cells on rapamycin and observed TOR1 hyperactivation, which leads to Hsp90-dependent calcineurin stabilization and thereby increased azole resistance. This in vitro finding was supported by in vivo data from a mouse model of systemic infection in which NKKY101 cells led to higher fungal load after fluconazole challenge than wild-type cells. Taken together, our study uncovers a mechanism of azole resistance in C. albicans, involving increased membrane rigidity and TOR signaling. [ABSTRACT FROM AUTHOR]