Features of Pro-K Important for Cleavage by SpoIVFB, an

Intramembrane proteases regulate diverse processes by cleaving substrates within a transmembrane segment or near the mem-brane surface. Bacillus subtilis SpoIVFB is an intramembrane metalloprotease that cleaves Pro-K during sporulation. To eluci-date features of Pro-K important for cleavage by SpoIVFB, coexpression of the two proteins in Escherichia coliwas used along with cell fractionation. In the absence of SpoIVFB, a portion of the Pro-K was peripherally membrane associated. This portion was not observed in the presence of SpoIVFB, suggesting that it serves as the substrate. Deletion of Pro-K residues 2 to 8, addi-tion of residues at its N terminus, or certain single-residue substitutions near the cleavage site impaired cleavage. Certain multi-residue substitutions near the cleavage site changed the position of cleavage, revealing preferences for a small residue preceding the cleavage site N-terminally (i.e., at the P1 position) and a hydrophobic residue at the second position following the cleavage site C-terminally (i.e., P2=). These features appear to be conserved among Pro-K orthologs. SpoIVFB did not tolerate an aro-matic residue at P1 or P2 = of Pro-K. A Lys residue at P3 = of Pro-K could not be replaced with Ala unless a Lys was provided farther C-terminally (e.g., at P9=).-Helix-destabilizing residues near the cleavage site were not crucial for SpoIVFB to cleave Pro-K. The preferences and tolerances of SpoIVFB are somewhat different from those of other intramembrane metallopro-teases, perhaps reflecting differences in the interaction of the substrate with the membrane and the enzyme. Regulated intramembrane proteolysis (RIP) was discovered alittle more than 10 years ago and has rapidly become a subject of intense investigation (1). RIP governs diverse processes tha