The prosequence of pro-sigmaK promotes membrane association and inhibits RNA polymerase core binding.
Pro-sigmaK is the inactive precursor of sigmaK, a mother cell-specific sigma factor responsible for the transcription of late sporulation genes of Bacillus subtilis. Upon subcellular fractionation, the majority of the pro-sigmaK was present in the membrane fraction. The rest of the pro-sigmaK was in a large complex that did not contain RNA polymerase core subunits. In contrast, the majority of the sigmaK was associated with core RNA polymerase. Virtually identical fractionation properties were observed when pro-sigmaE was analyzed. Pro-sigmaK 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-sigmaK did not require spoIVF gene products, which appear to be located in the mother cell membrane that surrounds the forespore, and govern pro-sigmaK processing in the mother cell. Furthermore, pro-sigmaK associated with the membrane when overproduced in vegetative cells. Overproduction of pro-sigmaK in sporulating cells resulted in more pro-sigmaK in the membrane fraction. In agreement with the results of cell fractionation experiments, immunofluorescence microscopy showed that pro-sigmaK 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-sigmaK. Treatment of extracts with 0.6 M KCl appeared to free most of the pro-sigmaK and sigmaK from other cell constituents. After salt removal, sigmaK, but not pro-sigmaK, reassociated with exogenous core RNA polymerase to form holoenzyme. These results suggest that the prosequence inhibits RNA polymerase core binding and targets pro-sigmaK to the membrane, where it may interact with the processing machinery.
Zhang
B
B
Department of Biochemistry, Michigan State University, East Lansing 48824, USA.
Hofmeister
A
A
Kroos
L
L
D001412:Bacillus subtilis / Q000502:physiology*; D001426:Bacterial Proteins; D002451:Cell Compartmentation; D002458:Cell Fractionation; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D008565:Membrane Proteins / Q000378:metabolism*; D008566:Membranes / Q000187:drug effects; D008954:Models, Biological; D017830:Octoxynol / Q000494:pharmacology; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000378:metabolism*; D012808:Sigma Factor / Q000378:metabolism*; D015398:Signal Transduction; D013171:Spores, Bacterial / Q000502:physiology; D014157:Transcription Factors / Q000378:metabolism*
D001426:Bacterial Proteins; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D012808:Sigma Factor; D014157:Transcription Factors; C112176:pro-sigmaK protein, Bacillus subtilis; C084190:sigma K; D017830:Octoxynol; D012321:DNA-Directed RNA Polymerases