title: Altered Escherichia coli membrane protein assembly machinery allows proper membrane assembly of eukaryotic protein vitamin K epoxide reductase.
abstract: Functional overexpression of polytopic membrane proteins, particularly when in a foreign host, is often a challenging task. Factors that negatively affect such processes are poorly understood. Using the mammalian membrane protein vitamin K epoxide reductase (VKORc1) as a reporter, we describe a genetic selection approach allowing the isolation of Escherichia coli mutants capable of functionally expressing this blood-coagulation enzyme. The isolated mutants map to components of membrane protein assembly and quality control proteins YidC and HslV. We show that changes in the VKORc1 sequence and in the YidC hydrophilic groove along with the inactivation of HslV promote VKORc1 activity and dramatically increase its expression level. We hypothesize that such changes correct for mismatches in the membrane topogenic signals between E. coli and eukaryotic cells guiding proper membrane integration. Furthermore, the obtained mutants allow the study of VKORc1 reaction mechanisms, inhibition by warfarin, and the high-throughput screening for potential anticoagulants.
authors.affiliation: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115;
authors.forename: Feras
authors.initials: F
authors.lastname: Hatahet
authors.affiliation: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115;
authors.forename: Jessica L
authors.initials: JL
authors.lastname: Blazyk
authors.affiliation: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115;
authors.forename: Eugenie
authors.initials: E
authors.lastname: Martineau
authors.affiliation: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115;
authors.forename: Eric
authors.initials: E
authors.lastname: Mandela
authors.affiliation: Department of Chemistry, University of Alberta, Edmonton, AB T6G 2R3, Canada.
authors.forename: Yongxin
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Department of Chemistry, University of Alberta, Edmonton, AB T6G 2R3, Canada.
authors.forename: Robert E
authors.initials: RE
authors.lastname: Campbell
authors.affiliation: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115; jon_beckwith@hms.harvard.edu.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Beckwith
authors.affiliation: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115;
authors.forename: Dana
authors.initials: D
authors.lastname: Boyd
mesh_terms: D000818:Animals; D002876:Chromosomes, Bacterial; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D009154:Mutation; D051381:Rats; D064417:Vitamin K Epoxide Reductases / Q000235:genetics / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D064417:Vitamin K Epoxide Reductases
keywords: VKORc1; YidC; anticoagulants; membrane protein assembly