Abstract
Signal peptidase is the enzyme responsible for removing the signal peptide portion of nascent presecretory and presumably also prelysosomal and premembrane proteins during their cotranslational translocation across the rough-endoplasmic reticulum (RER) membrane. This chapter describes the quantitative assay for signal peptidase that involves the translation of mRNA encoding a secretory protein in a cell-free in vitro translation system containing microsomal membranes. In this assay, signal peptidase activity is coupled to both the translation and translocation of the presecretory protein. [35S]Prolactin produced by posttranslational cleavage of [35S]preprolactin, in an assay containing 1.0% Triton X-100 and 2.5 mM phospholipid, is detected by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS) followed by autoradiography and is quantitated by counting the portion of the gel containing the prolactin band or by densitometry of the autoradiograph. Utilizing a qualitative assay for signal peptidase, signal peptidase can be inactivated by delipidation, and activity can be restored to the delipidated enzyme by readdition of phospholipid.
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