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 wit h the pro-sigma(K) processing complex, comprising SpoIVFA, BofA, and the pr o-sigma(K) processing protease, SpoIVFB, Mature sigma(K) then directs late gene expression in the parental compartment of the developing sporangial ce ll, The nature of this complex and how it is activated to process pro-sigma (K) are not understood. AII three proteins are predicted to be integral mem brane proteins. Here, we have analyzed the membrane topology of SpoIVFA and SpoIVFB by constructing chimeric forms of sponIVFA and spoIVFB with the co mplementary reporters phoA and lacZ and analyzing activity in Escherichia c oli, SpoIVFA was found to have a single transmembrane-spanning domain, whil e SpoIVFB was shown to have six transmembrane-spanning domains (6-transmemb rane configuration). Further, SpoIVFA is required to stabilize SpoIVFB in t he 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 ass embly of both SpoIVFA and SpoIVFB. The single transmembrane domain of SpoIV FA (approximately residues 73 to 90) was shown to be the principle determin ant in stabilizing the 6-transmembrane configuration of SpoIVFB, Although t he bofB8 allele, which uncouples the sigma(K) checkpoint, did not appear to promote a conformational change from a 6- to il-transmembrane configuratio n of SpoIVFB (apparently ruling out a profound conformational change as the mechanism of activating SpoIVFB proteolytic activity), instability of SpoI VFB may be an important factor in SpoTVFB-mediated processing of pro-sigma( K).