期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：49
页码：15184-15189
DOI：10.1073/pnas.1521260112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceProteins embedded in biological membranes are important targets for therapeutic intervention. However, they tend to be far more challenging to deal with than soluble proteins. We have used the anticoagulant target VKORc1 to investigate membrane protein expression bottlenecks in the bacterium Escherichia coli. When this poorly expressed enzyme is mutagenized or introduced to a library of E. coli mutants, functional expression is achieved. This approach gave clues on why certain foreign membrane proteins fail to correctly assimilate in E. coli membranes including mis-matches in membrane protein assembly signals. We also identified key critical residues in two components of membrane protein assembly and quality control for rational manipulations to enhance functional expression. Finally, we demonstrate the high-throughput screening for potential anticoagulants using bacteria. 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.
关键词：membrane protein assembly ; YidC ; VKORc1 ; anticoagulants
