1. DNA‐anchored LacI–CFP–Luciferase aggregates capture misfolded cytosolic proteins.
DNA‐anchored LacI–CFP–Luciferase aggregates capture misfolded cytosolic proteins. (A) LacI–CFP–Luciferase was produced in AB1157 cells harbouring multiple copies of lac operators at the terminus region (ter) of the bacterial chromosome. The localization of LacI–CFP–Luciferase was monitored at 25°C and after heat stress (45°C). The images show an overlay of the CFP signal (blue) and the membrane stain FM4‐64 (red). Numbers and positionings of LacI–CFP–Luciferase foci were determined at both temperatures (n=100, right panels). Scale bar: 1 μm. (B) MetA–YFP and LacI–CFP–Luciferase were co‐produced in AB1157 cells and images of the respective fusion protein were recorded at 30°C and after heat shock (45°C, 20 min). The overlay (merge) of both images reveals co‐aggregation of both model proteins at the ter region on heat stress. Membranes were stained with FM4‐64. Scale bar: 1 μm. (C) ClpB–YFP and LacI–CFP–Luciferase were co‐produced in AB1157 cells. Images of the respective fusion proteins were recorded prior (25°C) and after heat shock (45°C, 20 min), revealing re‐localization of ClpB–YFP to the ter region of the chromosome (see merge). (D) The localizations of stress‐induced MetA–YFP or ClpB–YFP foci, generated on co‐production of LacI–CFP–Luciferase in AB1157 cells, were quantified (n=100). The positioning of protein aggregates is indicated (white dot: co‐localization; yellow dot: exclusive MetA–YFP or ClpB–YFP localization).
Juliane Winkler et al. EMBO J. 2010;29:
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