Membrane topology of the Bacillus subtilis pro-sigma(K) processing complex.
Activation of the final sporulation-specific transcription factor, sigma(K), is regulated by a signal emanating from the forespore which interacts with the pro-sigma(K) processing complex, comprising SpoIVFA, BofA, and the pro-sigma(K) processing protease, SpoIVFB. Mature sigma(K) then directs late gene expression in the parental compartment of the developing sporangial cell. The nature of this complex and how it is activated to process pro-sigma(K) are not understood. All three proteins are predicted to be integral membrane proteins. Here, we have analyzed the membrane topology of SpoIVFA and SpoIVFB by constructing chimeric forms of spoIVFA and spoIVFB with the complementary reporters phoA and lacZ and analyzing activity in Escherichia coli. SpoIVFA was found to have a single transmembrane-spanning domain, while SpoIVFB was shown to have six transmembrane-spanning domains (6-transmembrane configuration). Further, SpoIVFA is required to stabilize SpoIVFB in the membrane. SpoIVFB was shown to have a 4-transmembrane configuration when expressed on its own but was found to have a 6-transmembrane configuration when coexpressed with SpoIVFA, while BofA had a positive effect on the assembly of both SpoIVFA and SpoIVFB. The single transmembrane domain of SpoIVFA (approximately residues 73 to 90) was shown to be the principle determinant in stabilizing the 6-transmembrane configuration of SpoIVFB. Although the bofB8 allele, which uncouples the sigma(K) checkpoint, did not appear to promote a conformational change from a 6- to 4-transmembrane configuration of SpoIVFB (apparently ruling out a profound conformational change as the mechanism of activating SpoIVFB proteolytic activity), instability of SpoIVFB may be an important factor in SpoIVFB-mediated processing of pro-sigma(K).
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School of Biological Sciences, Royal Holloway University of London, Egham, Surrey TW20 0EX, United Kingdom.
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D001412:Bacillus subtilis / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002462:Cell Membrane / Q000737:chemistry / Q000648:ultrastructure; D010450:Endopeptidases; D008565:Membrane Proteins / Q000737:chemistry; D011487:Protein Conformation; D011498:Protein Precursors / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012097:Repressor Proteins; D012808:Sigma Factor / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000378:metabolism*
D001426:Bacterial Proteins; D008565:Membrane Proteins; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D012808:Sigma Factor; D014157:Transcription Factors; C112176:pro-sigmaK protein, Bacillus subtilis; C074423:bofA protein, Bacillus subtilis; C071441:spoIVFA protein, Bacillus subtilis; D010450:Endopeptidases; C071442:spoIVFB protein, Bacillus subtilis