A mutation of the H-loop selectively affects rhodamine transport by the yeast multidrug ABC transporter Pdr5

Ernst, Robert; Kueppers, Petra; Klein, Cornelia M.; Schwarzmueller, Tobias; Kuchler, Karl; Schmitt, Lutz

doi:10.1073/pnas.0800191105

Cited by 146Drug Transport and Resistance MechanismsATP Synthase and ATPases ResearchAntibiotic Resistance in Bacteria

A mutation of the H-loop selectively affects rhodamine transport by the yeast multidrug ABC transporter Pdr5

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Ernst, Robert, Kueppers, Petra, Klein, Cornelia M., Schwarzmueller, Tobias, Kuchler, Karl, Schmitt, Lutz·2008·Proceedings of the National Academy of Sciences

DOI 10.1073/pnas.0800191105PMC PMC2278221

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Abstract

The yeast ABC transporter Pdr5 plays a major role in drug resistance against a large number of structurally unrelated compounds. Although Pdr5 has been extensively studied, many important aspects regarding its molecular mechanisms remain unresolved. For example, a striking degeneration of conserved amino acid residues exists in the nucleotide binding domains (NBDs), but their functional relevance is unknown. Here, we performed in vivo and in vitro experiments to address the functional asymmetry of NBDs. It became evident by ATPase activity and drug transport studies that catalysis at only one of the two NBD composite sites is crucial for protein function. Furthermore, mutations of the proposed “catalytic carboxylate” (E1036) and the “catalytic dyad histidine” (H1068) were characterized. Although a mutation of the glutamate abolished ATPase activity and substrate transport, mutation of H1068 had no influence on ATP consumption. However, the H1068A mutation abolished rhodamine transport in vivo and in vitro , while leaving the transport of other substrates unaffected. By contrast to mammalian P-glycoprotein (P-gp), the ATPase activity of yeast Pdr5 is not stimulated by the addition of substrates, indicating that Pdr5 is an uncoupled ABC transporter that constantly hydrolyses ATP to ensure active substrate transport. Taken together, our data provide important insights into the molecular mechanism of Pdr5 and suggest that not solely the transmembrane domains dictate substrate selection.

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Authors: Ernst, R., Kueppers, P., Klein, C. M., Schwarzmueller, T., Kuchler, K., & Schmitt, L.
Affiliations: Heinrich Heine University Düsseldorf (DE)
Max Perutz Labs (AT)
Max F. Perutz Laboratories, Medical University Vienna, Campus Vienna Biocenter, Dr. Bohr-Gasse 9/2, A-1030 Vienna, Austria
ORCID: Ernst, Robert
Kuchler, Karl
Schmitt, Lutz
Venue: Proceedings of the National Academy of Sciences
Publisher: National Academy of Sciences
Published: 2008-04
Volume: 105
Issue: 13
Pages: 5069-5074
ISSN: 0027-8424 (ISSN-L)
1091-6490
DOI: 10.1073/pnas.0800191105
PMC: PMC2278221
OpenAlex: W1964828092
Cited by: 146
References: 41
Citations by year: 2026: 4 · 2025: 4 · 2024: 4 · 2023: 4 · 2022: 7 · 2021: 6 · 2020: 9 · 2019: 12 · 2018: 6 · 2017: 11 · 2016: 8 · 2015: 5 · 2014: 11 · 2013: 10 · 2012: 8
FWCI: 6.52
Type: journal article
Language: en
Open access: green
www.ncbi.nlm.nih.gov
Locations: Proceedings of the National Academy of Sciences (publishedVersion)
PubMed Central (publishedVersion)
PubMed (publishedVersion)
Topics: Health Sciences › Medicine › Oncology › Drug Transport and Resistance Mechanisms
Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › ATP Synthase and ATPases Research
Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Medicine › Antibiotic Resistance in Bacteria
Keywords: PDR5, ABC transporters, multidrug resistance, nucleotide-binding domain, ATPase activity, substrate selection
MeSH terms: Adenosine Triphosphatases, Cell Membrane, Hydrolysis, Mutation, Phenotype, Rhodamines, Saccharomyces cerevisiae, Gene Expression Regulation, Fungal, Sequence Alignment, Conserved Sequence, ATP-Binding Cassette Transporters, Amino Acid Motifs, Saccharomyces cerevisiae Proteins

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A mutation of the H-loop selectively affects rhodamine transport by the yeast multidrug ABC transporter Pdr5

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