title: 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.
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Lyle A
authors.initials: LA
authors.lastname: Simmons
authors.forename: Alan D
authors.initials: AD
authors.lastname: Grossman
authors.forename: Graham C
authors.initials: GC
authors.lastname: Walker
mesh_terms: D000251:Adenosine Triphosphatases / Q000032:analysis / Q000235:genetics; D001412:Bacillus subtilis / Q000737:chemistry / Q000201:enzymology*; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics; D002462:Cell Membrane / Q000737:chemistry; D003600:Cytosol / Q000737:chemistry*; D049071:Endopeptidase Clp / Q000032:analysis* / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006360:Heat-Shock Proteins / Q000032:analysis / Q000235:genetics; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D049070:Protease La / Q000032:analysis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D012808:Sigma Factor / Q000032:analysis / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; C087481:ClpC protein, Bacteria; D006360:Heat-Shock Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D012808:Sigma Factor; C485184:enhanced green fluorescent protein; C426143:lonB protein, bacillus subtilis; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D049070:Protease La; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases; C118340:ClpE protein, bacteria