Figure 2. Ms DnaK is required for nascent Firefly luciferase folding.

(A) A₆₀₀ and relative Luciferase activity of a Tet-DnaK strain constitutively expressing firefly luciferase (MGM6006). A₆₀₀ (plotted on left Y axis) for +DnaK indicated by closed circles/solid line and for −DnaK as open circles/dashed line. %RLUs (plotted on right Y axis), calculated as Counts per second (CPS) of −DnaK cultures divided by CPS of +DnaK cultures multiplied by 100, indicated by grey squares/solid line. Time indicated on X axis in hours. Each point is the mean of 3 independent cultures. Error bars indicate standard deviation of replicates. (B) Luciferase and Dnak-STII protein levels during DnaK depletion. Immunoblot of lysates prepared from one representative culture of Tet-DnaK Luciferase strain (MGM6006) shown in A. Top panel is probed for firefly luciferase, middle for StrepTagII, bottom for RNAPβ as a loading control. (C) Solubility of luciferase from DnaK depleted cultures. Soluble and pellet protein fractions of lysates prepared from DnaK replete (+ATc) and DnaK depleted (−ATc for 15 hours). Top panel is probed for firefly luciferase, bottom for StrepTagII. (D) Luciferase-mCitrine is diffuse in DnaK replete cells. Time-lapse microscopy of a Tet-DnaK strain constitutively expressing firefly luciferase fused to mCitrine (MGM6010) grown in the presence of ATc. (E) Luciferase-mCitrine forms foci in DnaK depleted cells. Time-lapse microscopy of a Tet-DnaK strain constitutively expressing firefly luciferase fused to mCitrine (MGM6010) grown in the absence of ATc. For (D) and (E), YFP images shown. White numbers indicate time in hours with respect to presence or absence of ATc and white bars indicate 2 µm. Exposure times were YFP 250 ms.