Three-dimensional Reconstruction of the Saccharomyces cerevisiae Multidrug Resistance Protein Pdr5p

• Published in: The Journal of biological chemistry 2003-04, Vol.278 (14), p.11995-11999
• Main Authors: Ferreira-Pereira, Antonio, Marco, Sergio, Decottignies, Annabelle, Nader, Joseph, Goffeau, André, Rigaud, Jean-Louis
• Format: Article
• Language: English
• Subjects: ATP-Binding Cassette Transporters - chemistry; ATP-Binding Cassette Transporters - genetics; ATP-Binding Cassette Transporters - ultrastructure; Cell Membrane - physiology; Crystallography, X-Ray; Drug Resistance, Multiple; Microscopy, Electron; Protein Structure, Quaternary; Protein Structure, Tertiary; Saccharomyces cerevisiae - chemistry; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - ultrastructure
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M212198200

Pdr5p, the major multidrug exporter in Saccharomyces cerevisiae , is a member of the ATP-binding cassette (ABC) superfamily. Pdr5p shares similar mechanisms of substrate recognition and transport with the human MDR1-Pgp, despite an inverted topology of transmembrane and ATP-binding domains. The hexahistidine-tagged Pdr5p multidrug transporter was highly overexpressed in yeast strains where other ABC genes have been deleted. After solubilization and purification, the 160-kDa recombinant Pdr5p has been reconstituted into a lipid bilayer. Controlled detergent removal from Pdr5p-lipid-detergent micelles allowed the production of peculiar square-shaped particles coexisting with liposomes and proteoliposomes. These particles having 11 nm in side were well suited for single particle analysis by electron microscopy. From such analysis, a computed volume has been determined at 25-Å resolution, giving insight into the structural organization of Pdr5p. Comparison with the reported structures of different bacterial ABC transporters was consistent with a dimeric organization of Pdr5p in the square particles. Each monomer was composed of three subregions corresponding to a membrane region of about 50 Å in height that joins two well separated protruding stalks of about 40 Å in height, ending each one with a cytoplasmic nucleotide-binding domain (NBD) lobe of about 50–60 Å in diameter. The three-dimensional reconstruction of Pdr5p revealed a close arrangement and a structural asymmetric organization of the two NBDs that appeared oriented perpendicularly within a monomer. The existence of different angular positions of the NBDs, with respect to the stalks, suggest rotational movements during the catalytic cycle.