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TitleAltered Escherichia coli membrane protein assembly machinery allows proper membrane assembly of eukaryotic protein vitamin K epoxide reductase.
Publication TypeJournal Article
Year of Publication2015
AuthorsHatahet, Feras, Jessica L. Blazyk, Eugenie Martineau, Eric Mandela, Yongxin Zhao, Robert E. Campbell, Jonathan Beckwith, and Dana Boyd
JournalProc Natl Acad Sci U S A
Volume112
Issue49
Pagination15184-9
Date Published2015 Dec 8
ISSN1091-6490
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.

DOI10.1073/pnas.1521260112
Alternate JournalProc. Natl. Acad. Sci. U.S.A.
PubMed ID26598701
PubMed Central IDPMC4679017