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.