Cysteine Residues in SpoIVFB are Required for Regulated Intramembrane Proteolysis of pro-ðk
Smith, Sean M.L., 1986-
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Regulated Intramembrane Proteolysis (RIP) is a cell signaling pathway that is common to all forms of life. In RIP, a transmembrane protease (I-CLiP) cleaves a membrane-tethered ð factor that drives specific gene transcription. In the starvation-induced endosporulation process of B. Subtilis, pro-ðk is activated by an S2P I-CLiP, SpoIVFB. Other groups have studied the molecular mechanisms of this process through cysteine modification experiments of the I-CLiPs. In order to conduct this type of experiment in SpoIVFB, we attempted to create a cysteine-free SpoIVFB by performing site-directed mutagenesis on plasmids transfected into E. coli DE3 competent cells. Our Western blot analyses showed that the cysteine residues were essential in SpoIVFB function, and that a cysteine-free SpoIVFB did not cleave pro-ðk. Our findings suggest that the cysteine residues are important in either substrate recognition or structural stability of SpoIVFB. Although other S2P proteases differ at these sites, they may be individually unique in order to recognize their substrate.