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Updated: Nov 23, 2025

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes
Published on: August 13, 2012
Functional Significance of Conserved Cysteines in the Extracellular Loops of the ATP Binding Cassette Transporter
Lyubomir Dimitrov Stanchev1,2, Magdalena Marek2, Feng Xian3
1Department of Molecular Biochemistry, Faculty of Chemistry and Biochemistry, Ruhr University Bochum, 44801 Bochum, Germany.
Abstract:
The pleiotropic drug resistance (PDR) transporter Pdr11p is expressed under anaerobic growth conditions at the plasma membrane of the yeast Saccharomyces cerevisiae, where it facilitates the uptake of exogenous sterols. Members of the fungal PDR family contain six conserved cysteines in their extracellular loops (ECL). For the functional analysis of these cysteine residues in Pdr11p, we generated a series of single cysteine-to-serine mutants. All mutant proteins expressed well and displayed robust ATPase activity upon purification. Mass-spectrometry analysis identified two cysteine residues (C582 and C603) in ECL3 forming a disulfide bond. Further characterization by cell-based assays showed that all mutants are compromised in facilitating sterol uptake, protein stability, and trafficking to the plasma membrane. Our data highlight the fundamental importance of all six extracellular cysteine residues for the functional integrity of Pdr11p and provide new structural insights into the PDR family of transporters.
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