Substrate requirements for regulated intramembrane proteolysis of Bacillus subtilis pro-sigmaK.
During sporulation of Bacillus subtilis, pro-sigmaK is activated by regulated intramembrane proteolysis (RIP) in response to a signal from the forespore. RIP of pro-sigmaK removes its prosequence (amino acids 1 to 20), releasing sigmaK from the outer forespore membrane into the mother cell cytoplasm, in a reaction catalyzed by SpoIVFB, a metalloprotease in the S2P family of intramembrane-cleaving proteases. The requirements for pro-sigmaK to serve as a substrate for RIP were investigated by producing C-terminally truncated pro-sigmaK fused at different points to the green fluorescent protein (GFP) or hexahistidine in sporulating B. subtilis or in Escherichia coli engineered to coexpress SpoIVFB. Nearly half of pro-sigmaK (amino acids 1 to 117), including part of sigma factor region 2.4, was required for RIP of pro-sigmaK-GFP chimeras in sporulating B. subtilis. Likewise, pro-sigmaK-hexahistidine chimeras demonstrated that the N-terminal 117 amino acids of pro-sigma(K) are sufficient for RIP, although the N-terminal 126 amino acids, which includes all of region 2.4, allowed much better accumulation of the chimeric protein in sporulating B. subtilis and more efficient processing by SpoIVFB in E. coli. In contrast to the requirements for RIP, a much smaller N-terminal segment (amino acids 1 to 27) was sufficient for membrane localization of a pro-sigmaK-GFP chimera. Addition or deletion of five amino acids near the N terminus allowed accurate processing of pro-sigmaK, ruling out a mechanism in which SpoIVFB measures the distance from the N terminus to the cleavage site. A charge reversal at position 13 (substituting glutamate for lysine) reduced accumulation of pro-sigmaK and prevented detectable RIP by SpoIVFB. These results elucidate substrate requirements for RIP of pro-sigmaK by SpoIVFB and may have implications for substrate recognition by other S2P family members.
Prince
Heather
H
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA.
Zhou
Ruanbao
R
Kroos
Lee
L
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001412:Bacillus subtilis / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism; D049452:Green Fluorescent Proteins; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011487:Protein Conformation; D011498:Protein Precursors / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015183:Restriction Mapping; D012808:Sigma Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D012808:Sigma Factor; D014157:Transcription Factors; C112176:pro-sigmaK protein, Bacillus subtilis; D049452:Green Fluorescent Proteins