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, LepI) fused to green fluorescent protein (GFP) in the bacterium Escherichia coli, and compared this to overexpression of a soluble protein, GST-GFP. Proteomes of total lysates, purified aggregates and cytoplasmic membranes were analyzed by 1D- and 2D-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 2D 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; GroEL/S), and soluble proteases (HslUV, 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-pta pathway for ATP production and a down-regulated TCA 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.