Functional Significance of Conserved Cysteines in the Extracellular Loops of the ATP Binding Cassette Transporter Pdr11p.

Autor: Stanchev LD; Department of Molecular Biochemistry, Faculty of Chemistry and Biochemistry, Ruhr University Bochum, 44801 Bochum, Germany.; Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg, Denmark., Marek M; Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg, Denmark., Xian F; Proteomics of Cellular Signaling, Luxembourg Institute of Health, Rue Thomas Edison 1A-B, L-1445 Strassen, Luxembourg., Klöhn M; Department of Molecular Biochemistry, Faculty of Chemistry and Biochemistry, Ruhr University Bochum, 44801 Bochum, Germany., Silvestro D; Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg, Denmark., Dittmar G; Proteomics of Cellular Signaling, Luxembourg Institute of Health, Rue Thomas Edison 1A-B, L-1445 Strassen, Luxembourg., López-Marqués RL; Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg, Denmark., Günther Pomorski T; Department of Molecular Biochemistry, Faculty of Chemistry and Biochemistry, Ruhr University Bochum, 44801 Bochum, Germany.; Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg, Denmark.
Jazyk: angličtina
Zdroj: Journal of fungi (Basel, Switzerland) [J Fungi (Basel)] 2020 Dec 22; Vol. 7 (1). Date of Electronic Publication: 2020 Dec 22.
DOI: 10.3390/jof7010002
Abstrakt: 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.
Databáze: MEDLINE
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