Cytoplasmic degradation of ssrA-tagged proteins.
Degradation of ssrA-tagged proteins is a central feature of protein-quality control in all bacteria. In Escherichia coli, the ATP-dependent ClpXP and ClpAP proteases are thought to participate in this process, but their relative contributions to degradation of ssrA-tagged proteins in vivo have been uncertain because two adaptor proteins, ClpS and SspB, can modulate proteolysis of these substrates. Here, intracellular levels of these protease components and adaptors were determined during exponential growth and as cells entered early stationary phase. Levels of ClpA and ClpP increased about threefold during this transition, whereas ClpX, ClpS and SspB levels remained nearly constant. Using GFP-ssrA expressed from the chromosome as a degradation reporter, the effects of altered concentrations of different protease components or adaptor proteins were explored. Both ClpXP and ClpAP degraded GFP-ssrA in the cell, demonstrating that wild-type levels of SspB and ClpS do not inhibit ClpAP completely. Upon entry into stationary phase, increased levels of ClpAP resulted in increased degradation of ssrA-tagged substrates. As measured by maximum turnover rates, ClpXP degradation of GFP-ssrA in vivo was significantly more efficient than in vitro. Surprisingly, ClpX-dependent ClpP-independent degradation of GFP-ssrA was also observed. Thus, unfolding of this substrate by ClpX appears to enhance intracellular degradation by other proteases.
Farrell
Christopher M
CM
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Grossman
Alan D
AD
Sauer
Robert T
RT
D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D003470:Culture Media; D003593:Cytoplasm / Q000378:metabolism*; D049071:Endopeptidase Clp / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D018832:Molecular Chaperones / Q000378:metabolism*; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism*
D003470:Culture Media; D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; D012329:RNA, Bacterial; C096222:tmRNA; D049452:Green Fluorescent Proteins; C483568:ClpA protease, E coli; C483572:ClpP protease, E coli; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases; C088072:ClpX protein, E coli; D000074183:ATPases Associated with Diverse Cellular Activities