The ATP-dependent HslVU/ClpQY protease participates in turnover of cell division inhibitor SulA in Escherichia coli

Abstract

Escherichia coli mutants lacking activities of all known cytosolic ATP-dependent proteases (Lon, ClpAP, ClpXP, and HslVU), due to double deletions [DeltahslVU and Delta(clpPX-lon)], cannot grow at low (30 degrees C) or very high (45 degrees C) temperatures, unlike those carrying either of the deletions. Such growth defects were particularly marked when the deletions were introduced into strain MG1655 or W3110. To examine the functions of HslVU and other proteases further, revertants that can grow at 30 degrees C were isolated from the multiple-protease mutant and characterized. The revertants were found to carry a suppressor affecting either ftsZ (encoding a key cell division protein) or sulA (encoding the SulA inhibitor, which binds and inhibits FtsZ). Whereas the ftsZ mutations were identical to a mutation known to produce a protein refractory to SulA inhibition, the sulA mutations affected the promoter-operator region, reducing synthesis of SulA. These results suggested that the growth defect of the parental double-deletion mutant at a low temperature was due to the accumulation of excess SulA without DNA-damaging treatment. Consistent with these results, SulA in the double-deletion mutant was much more stable than that in the Delta(clpPX-lon) mutant, suggesting that SulA can be degraded by HslVU. As expected, purified HslVU protease degraded SulA (fused to the maltose-binding protein) efficiently in an ATP-dependent manner. These results suggest that HslVU as well as Lon participates in the in vivo turnover of SulA and that HslVU becomes essential for growth when the Lon (and Clp) protease level is reduced below a critical threshold.

FIG. 1

Nucleotide (or amino acid) alterations caused by suppressor mutations isolated in this study. (A) The ftsZ2691 allele of a class I revertant contained a 6-bp insertion near the 5′ end of the ftsZ coding region: TCGGCG (underlined) was repeated three times, instead of twice in the wild type. Numbers represent amino acid residues, starting from the N-terminal methionine (not shown). (B) All four class II revertants tested contained a mutation within the promoter-operator region of sulA. Arrows pointing up indicate mutational changes observed, and numbers in parentheses indicate independent revertants. The transcription start sites (6) are indicated by arrows pointing to the right; the first base of the longer transcript is base 1.

FIG. 2

Immunoblotting of SulA in the representative protease mutants studied, with or without suppressors. (A) Cells were grown in L broth at 42 or 30°C to the mid-log phase, and whole-cell proteins were prepared and analyzed by SDS-PAGE (13% polyacrylamide gel) followed by immunoblotting. (B) Cells were grown in L broth at 42°C and treated with mitomycin C. Samples were taken before (−) and 30 min after (+) the addition of mitomycin C. Whole-cell proteins were prepared and analyzed as described for panel A. Asterisks indicate a nonspecific band immediately below SulA. MG1655, wild type; KY2966, ΔhslVU; KY2347, Δ(clpPX-lon); KY2350, ΔhslVU Δ(clpPX-lon); KY2691 and KY2350, ftsZ2691; KY2981 and KY2350, sulA2981.

FIG. 3

Stability of mitomycin C-induced SulA in Δ(clpPX-lon) and ΔhslVU Δ(clpPX-lon) mutants. (A) Time course of accumulation of SulA upon addition of mitomycin C. Cells were grown in L broth at 42°C, and mitomycin C was added at time zero. Samples were taken at intervals, and whole-cell proteins were analyzed as described in the legend to Fig. 2A. (B) Stability of mitomycin C-induced SulA. Cells were grown in L broth at 42°C and treated with mitomycin C for 30 min, and spectinomycin (1 mg/ml) was added at time zero. Samples were taken at the times indicated, and the remaining SulA level was determined by immunoblotting. KY2347, Δ(clpPX-lon); KY2350, ΔhslVU Δ(clpPX-lon).

FIG. 4

Cellular levels of SulA, Lon, and ς³² upon mitomycin C treatment of the wild type (MG1655) and the ΔhslVU mutant (KY2966). Cells were grown in L broth at 42°C, mitomycin C was added, and samples taken at intervals were analyzed by SDS-PAGE and immunoblotting as described in the legend to Fig. 2A. Asterisks indicate a nonspecific band.

FIG. 5

In vitro degradation of SulA by purified HslVU protease. (A) Purified HslV (0.96 μg), HslU (2.4 μg), and MBP-SulA (2.4 μg) were mixed in a reaction mixture (60 μl) with or without 4 mM ATP as described in Materials and Methods. Samples were analyzed by SDS-PAGE before (lanes 1, 3, 5, and 7) or after (lanes 2, 4, 6, and 8) incubation at 37°C for 2 h. (B) Time course of degradation. HslV, HslU, MBP-SulA, and MBP-LacZα (2.4 μg) were mixed essentially as described for panel A and incubated at 37°C in the presence of 4 mM ATP. Samples were withdrawn at the indicated times before (0 min) and after incubation at 37°C and analyzed by SDS-PAGE.