Fig. 3: Mycobacterial proteins are differentially susceptible to degradation by ClpC1-FRB.

a Schematic of high-throughput flow cytometry screen in Msm cells expressing targets tagged with FKBP-eGFP. b Fluorescence of live cells as a proxy for protein levels of RpoA-FKBP-eGFP in the clpC1-frb background over time. c Summary plot of selected target proteins; fluorescence of live cells as a proxy for protein levels in the clpC1-frb background over time. Density matched log phase cells incubated with DMSO or 0.1 μg·mL⁻¹ rapamycin with shaking at 37 °C for the indicated times. The flow cytometry fluorescence distribution and degradation kinetics of the full set of target proteins are included in Supplementary Figs. 7c, d. d Enumeration of selected target proteins and their respective rate constants (dG/dt) and proportion of total degradation (% deg). The corresponding statistics for the full set of 54 protein targets are included in Supplementary Data File 2. e Correlation plot relating the log₂-transformed degradation rate and the steady state intensity of the 54 target proteins listed in Supplementary Data File 2. f Live cell, wide-field fluorescence microscopy images of cells expressing selected target proteins following 9 h of incubation with DMSO or 0.1 μg·mL⁻¹ rapamycin. Scale bar, 5 μm. For (b), data are two technical replicates, are representative of three independent experiments, and normalized to the mode; For (c), data are the mean of two technical replicates, are representative of two independent experiments, and normalized to fluorescent intensity of DMSO-treated cells at time = 0 h. The centerline in (e) denotes the linear fit between the measured steady state fluorescence intensity and the estimated degradation constant (log₂-transformed) and is bounded by the 95% confidence interval. The correlation coefficient (r) and the P-value in (e) were determined using a two-sided Pearson’s correlation test. Data in (f) are representative microscopy images selected from among 12 fields for each strain and are representative of two independent experiments. a Created with BioRender.com, released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license. Source data are provided with this paper.