Clp and Lon proteases occupy distinct subcellular positions in Bacillus subtilis

Abstract

Among other functions, ATP-dependent proteases degrade misfolded proteins and remove several key regulatory proteins necessary to activate stress responses. In Bacillus subtilis, ClpX, ClpE, and ClpC form homohexameric ATPases that couple to the ClpP peptidase. To understand where these peptidases and ATPases localize in living cells, each protein was fused to a fluorescent moiety. We found that ClpX-GFP (green fluorescent protein) and ClpP-GFP localized as focal assemblies in areas that were not occupied by the nucleoid. We found that the percentage of cells with ClpP-GFP foci increased following heat shock independently of protein synthesis. We determined that ClpE-YFP (yellow fluorescent protein) and ClpC-YFP formed foci coincident with nucleoid edges, usually near cell poles. Furthermore, we found that ClpQ-YFP (HslV) localized as small foci, usually positioned near the cell membrane. We found that ClpQ-YFP foci were dependent on the presence of the cognate hexameric ATPase ClpY (HslU). Moreover, we found that LonA-GFP is coincident with the nucleoid during normal growth and that LonA-GFP also localized to the forespore during development. We also investigated LonB-GFP and found that this protein localized to the forespore membrane early in development, followed by localization throughout the forespore later in development. Our comprehensive study has shown that in B. subtilis several ATP-fueled proteases occupy distinct subcellular locations. With these data, we suggest that substrate specificity could be determined, in part, by the spatial and temporal organization of proteases in vivo.

FIG. 1.

ClpX-GFP and ClpP-GFP form foci in nucleoid-excluded areas. (A) Positions of single ClpX-GFP foci scored from cell pole to focus and plotted relative to cell length. (B) Positions of single ClpP-GFP foci scored from cell pole to focus and plotted relative to cell length. For each plot, the left vertical axis denotes the near cell pole. The solid lines mark the midcell and the other cell pole, respectively. The dashed lines mark the cell quarter positions. The number of cells scored is indicated, and the focus scoring was performed as described previously (2, 68). (C) ClpP-GFP (green) with vital membrane stain FM4-64 (red); (D) ClpP-GFP with membrane stain following temperature shift for 20 min to 42°C. (E) ClpX-GFP with membrane; (F) ClpX-GFP with membrane following temperature shift for 20 min to 42°C. (G) ClpP-GFP at 30°C after 20 min; (H) ClpP-GFP at 42°C for 20 min; (I) ClpP-GFP pretreated with chloramphenicol prior to a shift to 42°C for 20 min. (J) ClpP-GFP foci (white) and (K) the corresponding nucleoid stained with DAPI (blue) are shown after treatment with chloramphenicol and heat shock at 42°C for 20 min; (L) merge of ClpP-GFP (white) and the nucleoid stained with DAPI (blue). Bar, 2 μm.

FIG. 2.

ClpX-YFP and ClpP-CFP foci colocalize during normal growth and following heat shock. (A) ClpX-YFP; (B) ClpP-CFP; (C) merge of ClpX-YFP and ClpP-CFP; (D) membrane stained with FM4-64. (E) ClpX-YFP at 42°C; (F) ClpP-CFP at 42°C; (G) merge of ClpX-YFP and ClpP-CFP; (H) corresponding membrane stain. (I) Quantitation of the percentage of ClpP-CFP foci paired with ClpX-YFP or free (unpaired) with ClpX. Arrows in panel G highlight free ClpP-CFP foci. The percentage of cells with ClpP-CFP was ∼98% (n = 414) with foci following heat treatment. For the ClpX-YFP images shown in panels A and E, the 90-ms exposure does not capture the diffuse fluorescence that ClpX-YFP forms. Bar, 2 μm.

FIG. 3.

ClpE-YFP and ClpC-YFP form foci on nucleoid edges near cell poles. (A) Positions of single ClpE-YFP foci scored from cell pole to focus and plotted relative to cell length. (B) Positions of single ClpC-YFP foci scored from cell pole to focus and plotted relative to cell length. For each plot, the left vertical axis denotes the near cell pole. The solid lines mark the midcell and other cell pole, respectively. The dashed lines mark the cell quarter positions. The number of cells scored is indicated in the graph. (C) ClpE-YFP; (D) corresponding DAPI. (E) ClpC-YFP; (F) corresponding DAPI. (G) ClpE-YFP (green) and membrane (red). (H) ClpC-YFP (green) and membrane (red). The exposure time for ClpE-YFP and ClpC-YFP was 2 s. The cell membrane was stained with FM4-64. Bar, 2 μm.

FIG. 4.

ClpQ-YFP is distributed diffusely and forms small foci. (A) Homology model of the B. subtilis ClpQ (HslV) monomer. The residues colored in red represent the C-terminal 8 amino acids of ClpQ (DQIILEEL). Shown is a side view. The “surface” represents the outside and the solvent-exposed exterior of the proteolytic chamber, and the “chamber” represents the site of peptide proteolysis when assembled into a dodecamer. (B) ClpQ-YFP and membrane in a clpY mutant background; (C) ClpQ-YFP in a clpY⁺ background; (D) merge of ClpQ-YFP and membrane (red). (E) Corresponding DNA (blue) and membrane (red) for ClpQ-YFP; (F) merge of ClpQ-YFP with DNA and membrane. Arrows highlight the positions of several ClpQ-YFP foci. The membrane was stained with FM4-64. Bar, 2 μm.

FIG. 5.

LonA-GFP localizes to the nucleoid and the forespore. (A) LonA-GFP and membrane; (B) corresponding DNA (DAPI) and membrane. (C) LonA-GFP at 42°C and membrane; (D) corresponding DNA (DAPI) and membrane. (E) LonA-GFP differential interference contrast (DIC); (F) corresponding LonA-GFP. (G) LonB-GFP; (H) corresponding membrane; (I) merge of LonB-GFP with membrane. (J) DIC corresponding to LonB-GFP; (K) LonB-GFP. The time following induction of sporulation is indicated in the appropriate panels. The membrane was stained with FM4-64. Bar, 2 μm.

FIG. 6.

Distributions of ClpP-GFP, ClpX-GFP, ClpE-YFP, and ClpC-YFP foci relative to the nucleoid. We scored the positions of the ClpP-GFP peptidase and each ClpP-associated ATPase in single cells relative to the nucleoid during normal growth in S7₅₀ defined minimal medium. (A) Description of the position of the focus. (B) Schematic representation of the subcellular localization described. The boundary of the cell is represented by a solid black line, the nucleoid is dashed, and the focus is represented by the shaded oval. (C) Percentages of cells with foci localized to the indicated subcellular positions. The number of cells scored is indicated below each column. It should be noted that when foci were scored to be located at “midcell” the foci that qualified were located between nucleoids and did not appear to be located on the nucleoid edge. In the case of ClpE-YFP and ClpC-YFP, a considerable number of foci were located at “midcell” but were on the edge of the nucleoid and were thus scored as “coincident with the nucleoid edge.” The experiments used to generate the data presented were independent from those used to generate the data presented in Table 2.