Release of signal peptide fragments into the cytosol requires cleavage in the transmembrane region by a protease activity that is specifically blocked by a novel cysteine protease inhibitor

Signal peptides of secretory and membrane proteins are generated by proteol ytic processing of precursor proteins after insertion into the endoplasmic reticulum membrane. Liberated signal peptides can be further processed, and the resulting N-terminal fragments are released toward the cytosol, where they may interact with target proteins like calmodulin. We show here that t he processing of signal peptides requires a protease activity distinct from signal peptidase, This activity is inhibited specifically with a newly dev eloped cysteine protease inhibitor, 1,3-di-(N-carboxybenzoyl-L-leucyl-L-leu cyl)amino acetone ((Z-LL)(2) ketone). Inhibitor studies revealed that the f inal, (Z-LL)(2) ketone sensitive cleavage event occurs within the hydrophob ic transmembrane region of the signal peptide, thus promoting the release o f an N-terminal fragment into the cytosol.