Fig. 3: Measurement of protein turnover in protease knockout strains enables proteome-wide identification of protease substrates.

A Scatter plots of protein total half-lives of N-limited wild type (WT) compared to ΔclpP, Δlon, and ΔhslV knockout (KO) cells. Dotted lines mark the dilution limit, the solid black line indicates perfect agreement. Substrates (black x) increase their total half-lives in KOs with high confidence (t-test, one-sided, p-value < 0.10). ClpA (pink), Tag (teal), and UhpA (purple) are the substrates of ΔclpP, Δlon, and ΔhslV, respectively. However, the protein YbhA (orange) is still degraded in individual KOs. Contour plots containing 50% of the probability mass for the cytoplasmic (red) and membrane (green) proteins indicate that individual KOs degrade bulk cytoplasmic proteins. B Since ΔftsH cells cannot grow in chemostats, we repeated the batch starvation assay as in Fig. 3E. The box extends from the first quartile to the third quartile, with a line at the median. The whiskers indicate 1.5× the inter-quartile range from the box. Results indicate that the ΔftsH cells, like the WT, also degrade their cytoplasmic proteins under nitrogen starvation (t-test, two-sided, p-value = 2E–12 for WT and p-value < 2E–16 for ΔftsH, n = 1519). C Scatter plots of protein total half-lives of WT and ΔclpP Δlon ΔhslV cells in N-lim. The substrates (black x) increase their total half-lives in the triple KO (t-test, one-sided, p-value < 0.09). Many proteins are still degrading in the triple KO, e.g., LexA and YoaC (blue). In fact, the bulk cytoplasm is still degraded. However, many more proteins are stabilized in the triple KO compared to the individual KOs, indicating redundancy among substrates, e.g., YbhA (orange). D Comparing the shifts in the WT and KO strains’ total half-lives, we assign each protease’s contribution to active protein turnover. The bar graph represents examples from each of the six categories—turnover explained predominantly by ClpP, Lon, HslV, additive contributions, redundant contributions, and actively degrading proteins in the triple KO. E Bar graph for the number of substrates and the % of the proteome assigned to each of the six categories described in (D). F Comparison of the substrates from our categories in E with previous proteome-wide substrate-protease assignment studies. ClpP trapped substrates significantly overlap with the identified ClpP substrates (Fisher test, two-sided, p-value = 6E–9), and previously identified substrates of HslV and FtsH show a significant overlap with redundant and additive substrates (Fisher test, two-sided, p-value = 1E–3). G Comparison of the percentage of active turnover per hour across the protease KOs under N-lim. Even after knocking out hslV, lon, and clpP simultaneously, 40% of the WT proteome turnover remains, suggesting that a major pathway of protein degradation in E. coli remains to be discovered.