Proteolysis has emerged as a central feature of physiology and gene control in cells of many kinds. We now know that the role of proteolysis is not restricted to protein quality control. Today, it is clear the cell uses multiple mechanisms, including proteolysis, to carefully regulate gene expression. Here, I report my studies on the ClpP proteases during growth and sporulation in the Gram-positive soil bacterium, B. subtilis. The ClpP proteases are highly conserved among bacteria and are also found within the chloroplasts and mitochondria of eukaryotes. Analogous to the cylindrical structure of the eukaryotic proteasome, the active sites of the ClpP proteases are compartmentalized and substrate selection is tightly controlled. The ClpP proteases are composed of two polypeptide types: the ClpP peptidase and a regulatory ATPase. In B. subtilis , there are three ATPases that can associate with the ClpP peptidase: ClpC, ClpE, or ClpX. The ATPase subunits bind, unfold, and translocate various substrates into the proteolytic compartment of ClpP, the spectrum of substrates degraded being determined by the ATPase subunit.
Here I report the use of the Green Fluorescent Protein to visualize the ClpP proteases during growth and sporulation. I show that during growth ClpC, ClpE, ClpX, and ClpP localize as foci near the cell poles and do so independently of one another. I further demonstrate that the region of ClpC containing the ATPase domain (AAA2) is necessary and sufficient for polar focus formation. I speculate that AAA2 is responsible for polar focus formation by ClpE and ClpX as well.
Sporulation involves the formation of an asymmetrically positioned septum that divides the developing cell into a smaller, prospective spore called the forespore and a larger, forespore-nurturing mother cell. I report that ClpC and ClpX differentially accumulate in the forespore and mother cell, respectively, during sporulation. Using model substrates for the two proteases, I showed that the differential accumulation of the ClpCP and ClpXP proteases results in differential proteolysis of their respective substrates. I speculate that the preferential action of ClpCP in the forespore contributes to the selective activation in the forespore of the transcription factor σ F by degradation of the anti-σF factor SpoIIAB. SpoIIAB is known to be degraded in a manner that depends on ClpCP and the C-terminal sequence LCN, which is necessary and sufficient for this degradation.
Efforts to demonstrate degradation of SpoIIAB in vitro and by heterologous production of ClpCP in E. coli were unsuccessful, leading to the hypothesis that ClpCP recognizes SpoIIAB by means of an unkown adaptor. I designed and executed a genetic screen to identify the adaptor based on the use of the Green Fluorescent Protein tagged with LCN at its C-terminus. I was able to isolate and map several mutants in which the level of fluorescence from the tagged protein was increased, but none were attractive candidates for an adaptor.
I conclude that the action of ClpP proteases in B. subtilis is governed both by subcellular localization and cell-specific accumulation.