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Journal of Bacteriology, October 2008, p. 6758-6768, Vol. 190, No. 20
0021-9193/08/$08.00+0     doi:10.1128/JB.00590-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Clp and Lon Proteases Occupy Distinct Subcellular Positions in Bacillus subtilis

Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts

Received 29 April 2008/ Accepted 31 July 2008

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.

Published ahead of print on 8 August 2008.

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