Experimental detection of proteolytic activity in a signal peptide peptidase of Arabidopsis thaliana

Signal peptide peptidase (SPP) is a multi-transmembrane aspartic protease involved in intramembrane-regulated proteolysis (RIP). RIP proteases mediate various key life events by releasing bioactive peptides from the plane of the membrane region. We have previously isolated Arabidopsis SPP (AtSPP) and found that this protein is expressed in the ER. An AtSPP-knockout plant was found to be lethal because of abnormal pollen formation; however, there is negligible information describing the physiological function of AtSPP. In this study, we have investigated the proteolytic activity of AtSPP to define the function of SPPs in plants.

The data from the in vitro cell-free assay indicated that the membrane fraction of both Arabidopsis cells and AtSPP recombinantly expressed in yeast actually possessed proteolytic activity for a human SPP substrate. We concluded that plant SPP possesses proteolytic activity and may be involved in RIP.

We found that an n-dodecyl-ß-maltoside (DDM)-solubilized membrane fraction from Arabidopsis cells digested the myc-Prolactin-PP-Flag peptide, a human SPP substrate, and this activity was inhibited by (Z-LL)2-ketone, an SPP-specific inhibitor. The proteolytic activities from the membrane fractions solubilized by other detergents were not inhibited by (Z-LL)2-ketone. To confirm the proteolytic activity of AtSPP, the protein was expressed as either a GFP fusion protein or solely AtSPP in yeast. SDS-PAGE analysis showed that migration of the fragments that were cleaved by AtSPP were identical in size to the fragments produced by human SPP using the same substrate. These membrane-expressed proteins digested the substrate in a manner similar to that in Arabidopsis cells. Conclusions: The data from the in vitro cell-free assay indicated that the membrane fraction of both Arabidopsis cells and AtSPP recombinantly expressed in yeast actually possessed proteolytic activity for a human SPP substrate. We concluded that plant SPP possesses proteolytic activity and may be involved in RIP.