FIGURE 8.

Model for the topology of Pen-2 and the interaction between Pen-2 and PS1. A, model for PS1 containing the catalytic site (18–21, 60). PS1 has 10 hydrophobic domains from which nine cross the membrane as transmembrane domains (TMDs, indicated by the bundle of numbered cylinders). The other one is present in the large loop between TMD6 and TMD7 and contains the endoproteolytic site (HDVII). In the figure, HDVII has undergone endoproteolysis and is divided in two parts. TMD6 and TMD7 delineate a water-containing cavity inside the membrane with the two catalytic aspartates facing each other. TMD9 and HDVII participate in the formation of this cavity as well. Water accessibility data of PS1 suggest that the catalytic cavity is open toward the cytosol (black line). Furthermore, an initial substrate-binding site has been proposed to allow lateral gating of the hydrophobic substrates into the hydrophilic catalytic cavity. TMD9 has been suggested to be part of this substrate binding site (pale line). Therefore, TMD9 is part of both the substrate-binding site and the catalytic site. Moreover, its flexible nature, induced by the PAL motif, is proposed to be important for the gating mechanism. B and C, model for the interaction of Pen-2 and PS1 as a drawn-open (B) and closed (C) picture. Hydrophobic domain 1 of Pen-2 (I) is supposed to interact with TMD4 of PS1 NTF (30, 31), whereas our cross-linking data position the loop of Pen-2 close to PS1 CTF. Furthermore, our water accessibility data of Pen-2 reveal that the loop of Pen-2 is accessible from the lumenal site. Similarly, the N-terminal part of TMD9 of PS1 containing the PAL motif, is accessible to impermeable sulfhydryl-reactive reagents as well and therefore exhibits a comparable accessibility pattern as the loop of Pen-2 (21). Therefore, we draw the loop of Pen-2 (red) in close proximity to PS1 TMD9. The N terminus of Pen-2 is present in a constricted environment indicated by the gray circles.