Localization of general and regulatory proteolysis in Bacillus subtilis cells

Abstract

Protein degradation mediated by ATP-dependent proteases, such as Hsp100/Clp and related AAA+ proteins, plays an important role in cellular protein homeostasis, protein quality control and the regulation of, e.g. heat shock adaptation and other cellular differentiation processes. ClpCP with its adaptor proteins and other related proteases, such as ClpXP or ClpEP of Bacillus subtilis, are involved in general and regulatory proteolysis. To determine if proteolysis occurs at specific locations in B. subtilis cells, we analysed the subcellular distribution of the Clp system together with adaptor and general and regulatory substrate proteins, under different environmental conditions. We can demonstrate that the ATPase and the proteolytic subunit of the Clp proteases, as well as the adaptor or substrate proteins, form visible foci, representing active protease clusters localized to the polar and to the mid-cell region. These clusters could represent a compartmentalized place for protein degradation positioned at the pole close to where most of the cellular protein biosynthesis and also protein quality control are taking place, thereby spatially separating protein synthesis and degradation.

Fig. 1

Subcellular localization of the Clp proteins. A. Upper panel: phase contrast images of B. subtilis strains encoding ClpP–GFP, ClpX–YFP and ClpC–GFP cultivated in LB medium at 30°C. Lower panel: overlay of the GFP signal of the fusion proteins (pseudocoloured in green) and the nucleoid stained with DAPI (pseudocoloured in red). B. Dynamic localization of ClpP–GFP. A selected field of the time-lapse movie (see Movie S2) with pictures taken every 8 min as indicated in the top left corner of the respective picture (See Experimental procedures for details).

Fig. 2

Localization of the Clp proteins upon heat schock. Cellular localization of ClpP–GFP, ClpC–GFP, ClpX–YFP and ClpE–YFP after heat shock at 50°C. The images show an overlay of the GFP/YFP signal of the fusion proteins (pseudocoloured in green) and the nucleoid stain DAPI (pseudocoloured in red).

Fig. 3

Colocalization of Clp proteases. Dual labelling of ClpX–ClpP (upper panel), ClpC–ClpP (middle panel) and ClpC–ClpX (lower panel) at 30°C. The images were obtained using YFP filters (left column) and CFP filters (middle column). An overlay of the YFP and CFP channels is shown on the right. YFP fluorescence has been pseudocoloured in red and CFP in green.

Fig. 4

Analysis of Clp proteins in different mutant strains. Cellular localization of ClpP–GFP in a ΔclpX background, ClpX–YFP in a ΔclpP background, ClpP–GFP in a ΔclpC background and ClpC–GFP in a ΔclpP background, before (30°C) and after heat shock (50°C).

Fig. 5

Analysis of Clp protease adaptor and substrate localization in different mutant strains. Cellular localization of adaptor McsB (McsB–YFP) in wild type, ΔclpC, ΔywlE or clpC-DWB background and substrate CtsR (CtsR–GFP) in wild type or a clpC-DWB background, before (30°C) or after heat shock (50°C).

Fig. 6

Colocalization of ClpP, ClpX, ClpC and ClpE with inclusion bodies. Inclusion bodies were visualized by phase contrast microscopy (left panel) and the different GFP/YFP fusions were visualized by fluorescence microscopy (right panel). Arrows indicate inclusion bodies visible in phase contrast images.

Fig. 7

Cellular localization of ClpP in the absence of cell division and replication. The ClpP localization is visualized in wild type (upper panel), and FtsZ-depleted B. subtilis strains at 30°C (middle panel), and in a DnaA-depleted B. subtilis strain at 50°C (lower panel). Phase contrast (i), membrane stain (ii), nucleoid stain (iii), GFP fluorescence (iv) and the merged images (v) are subsequently depicted (see Experimental procedures for details).