title: Consequences of membrane protein overexpression in Escherichia coli.
abstract: Overexpression of membrane proteins is often essential for structural and functional studies, but yields are frequently too low. An understanding of the physiological response to overexpression is needed to improve such yields. Therefore, we analyzed the consequences of overexpression of three different membrane proteins (YidC, YedZ, and LepI) fused to green fluorescent protein (GFP) in the bacterium Escherichia coli and compared this with overexpression of a soluble protein, GST-GFP. Proteomes of total lysates, purified aggregates, and cytoplasmic membranes were analyzed by one- and two-dimensional gel electrophoresis and mass spectrometry complemented with flow cytometry, microscopy, Western blotting, and pulse labeling experiments. Composition and accumulation levels of protein complexes in the cytoplasmic membrane were analyzed with improved two-dimensional blue native PAGE. Overexpression of the three membrane proteins, but not soluble GST-GFP, resulted in accumulation of cytoplasmic aggregates containing the overexpressed proteins, chaperones (DnaK/J and GroEL/S), and soluble proteases (HslUV and ClpXP) as well as many precursors of periplasmic and outer membrane proteins. This was consistent with lowered accumulation levels of secreted proteins in the three membrane protein overexpressors and is likely to be a direct consequence of saturation of the cytoplasmic membrane protein translocation machinery. Importantly accumulation levels of respiratory chain complexes in the cytoplasmic membrane were strongly reduced. Induction of the acetate-phosphotransacetylase pathway for ATP production and a down-regulated tricarboxylic acid cycle indicated the activation of the Arc two-component system, which mediates adaptive responses to changing respiratory states. This study provides a basis for designing rational strategies to improve yields of membrane protein overexpression in E. coli.
authors: Department of Biochemistry and Biophysics, Center for Biomembrane Research, Stockholm University, SE-106 91, Stockholm, Sweden.
authors: Samuel
authors: S
authors: Wagner
authors: Louise
authors: L
authors: Baars
authors: A Jimmy
authors: AJ
authors: Ytterberg
authors: Anja
authors: A
authors: Klussmeier
authors: Claudia S
authors: CS
authors: Wagner
authors: Olof
authors: O
authors: Nord
authors: Per-Ake
authors: PA
authors: Nygren
authors: Klaas J
authors: KJ
authors: van Wijk
authors: Jan-Willem
authors: JW
authors: de Gier
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D002462:Cell Membrane / Q000378:metabolism; D019295:Computational Biology / Q000379:methods; D003593:Cytoplasm / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry / Q000379:methods; D008565:Membrane Proteins / Q000737:chemistry*; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000378:metabolism; D021381:Protein Transport; D040901:Proteomics / Q000379:methods
chemical_list: D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate