The prosequence of Pro-σK promotes membrane association and inhibits RNA polymerase core binding

Pro-sigma(K) is the inactive precursor of sigma(K), a mother cell-specific sigma factor responsible for the transcrip of late sporulation genes of Bacillus subtilis, Upon subcellular fractionation, the majority of the pro-sigma(K) was present in the membrane fraction. The rest of the pro-sigma(K) was in a large complex that did not contain RNA polymerase core subunits, In contrast, the majority of the sigma(K) was associated with core RNA polymerase, Virtually identical fractionation properties were observed when pro-sigma(K) was analyzed. Pro-sigma(K) was completely solubilized from the membrane fraction and the large complex by Triton X-100 and was partially solubilized from the membrane fraction by NaCl and KSCN. The membrane association of pro-sigma(K) did not require spoIVF gene products, which appear to be located in the mother cell membrane that surrounds the forespore, and govern pro-sigma(K) processing in the mother cell, Furthermore, pro-sigma(K) associated with the membrane when overproduced in vegetative cells. Overproduction of pro-sigma(K) in sporulating cells resulted in more pro-sigma(K) in the membrane fraction. In agreement with the results of cell fractionation experiments, immunofluorescence microscopy showed that pro-sigma(K) was localized to the mother cell membranes that surround the mother cell and the forespore in sporulating wild-type cells and mutant cells that do not process pro-sigma(K). Treatment of extracts with 0.6 M KCl appeared to free most of the pro-sigma(K) and sigma(K) from other cell constituents. After salt removal, sigma(K), but not pro-sigma(K), reassociated with exogenous core RNA polymerase to form holoenzyme. These results suggest that the prosequence inhibits RNA polymerase core binding and targets pro-sigma(K) to the membrane, where it may interact with the processing machinery.