title: Genome-Wide Screen Revealssec21Mutants ofSaccharomyces cerevisiaeAre Methotrexate-Resistant.
abstract: Drug resistance is a consequence of how most modern medicines work. Drugs exert pressure on cells that causes death or the evolution of resistance. Indeed, highly specific drugs are rendered ineffective by a single DNA mutation. In this study, we apply the drug methotrexate, which is widely used in cancer and rheumatoid arthritis, and perform evolution experiments on Baker's yeast to ask the different ways in which cells become drug resistant. Because of the conserved nature of biological pathways between yeast and man, our results can inform how the same mechanism may operate to render human cells resistant to treatment. Exposure of cells to small molecules and drug therapies imposes a strong selective pressure. As a result, cells rapidly acquire mutations in order to survive. These include resistant variants of the drug target as well as those that modulate drug transport and detoxification. To systematically explore how cells acquire drug resistance in an unbiased manner, rapid cost-effective approaches are required. Methotrexate, as one of the first rationally designed anticancer drugs, has served as a prototypic example of such acquired resistance. Known methotrexate resistance mechanisms include mutations that increase expression of the dihydrofolate reductase (DHFR) target as well as those that maintain function yet reduce the drug's binding affinity. Recent evidence suggests that target-independent, epistatic mutations can also result in resistance to methotrexate. Currently, however, the relative contribution of such unlinked resistance mutations is not well understood. To address this issue, we took advantage ofSaccharomyces cerevisiaeas a model eukaryotic system that combined with whole-genome sequencing and a rapid screening methodology, allowed the identification of causative mutations that modulate resistance to methotrexate. We found a recurrent missense mutation inSEC21(orthologous to human COPG1), which we confirmed in 10de novomethotrexate-resistant strains. Thissec21allele (S96L) behaves as a recessive, gain-of-function allele, conferring methotrexate resistance that is abrogated by the presence of a wild-type copy ofSEC21These observations indicate that the Sec21p/COPI transport complex has previously uncharacterized roles in modulating methotrexate stress.
authors.affiliation: Department of Pharmaceutical Sciences, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
authors.forename: Lai H
authors.identifier: 0000-0001-8653-2734
authors.initials: LH
authors.lastname: Wong
authors.affiliation: Department of Pharmaceutical Sciences, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.|Department of Zoology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
authors.forename: Stephane
authors.initials: S
authors.lastname: Flibotte
authors.affiliation: Department of Pharmaceutical Sciences, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
authors.forename: Sunita
authors.initials: S
authors.lastname: Sinha
authors.affiliation: Department of Pharmaceutical Sciences, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Chiang
authors.affiliation: Department of Pharmaceutical Sciences, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
authors.forename: Guri
authors.initials: G
authors.lastname: Giaever
authors.affiliation: Department of Pharmaceutical Sciences, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada corey.nislow@ubc.ca.
authors.forename: Corey
authors.initials: C
authors.lastname: Nislow
mesh_terms: D025141:Drug Resistance, Fungal / Q000187:drug effects / Q000235:genetics*; D005816:Genetic Complementation Test; D014644:Genetic Variation; D016681:Genome, Fungal; D008727:Methotrexate / Q000494:pharmacology*; D009154:Mutation; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: C000627659:SEC21 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; D008727:Methotrexate
keywords: COPI complex; budding yeast