S4-01-06: Mechanisms of intramembrane proteolysis by gamma-secretase and related proteases

Abstract

Background: -Secretase is a promiscuous aspartyl protease responsible for the final intramembrane cleavage of various type I transmembrane proteins after their large ectodomains are shed. The vast functional diversity of its substrates, which are involved in cell fate decisions, adhesion, neurite outgrowth and synapse formation, highlights the important role -secretase plays in development, neurogenesis and neurodegeneration. The most renowned substrates are the amyloid precursor protein and Notch, from which -secretase liberates amyloid peptides and induces downstream signaling, respectively. -Secretase is a multiprotein complex containing presenilin -which harbours the catalytic site-, nicastrin, APH-1 and PEN-2. Despite the efforts, we are now only beginning to unravel the assembly, stoichiometry, activation and subcellular location of -secretase complexes. Given the multisubunit character of -secretase, we hypothesize that assembly must be regulated by additional factors early in the biosynthetic compartments. Methods: -Secretase complex assembly was studied in HeLa cells and wild-type or nicastrin-/-, presenilin-/and APH1-/mouse embryonic fibroblasts (MEF) using a variety of molecular biological approaches including site-directed mutagenesis, siRNA, ectopic overexpression and protein electrophoresis. Results: Our recent findings indeed support the working hypothesis that assembly of this complex occurs during ER-Golgi recycling and is at least mediated through interactions of nicastrin with the cargo retrieval receptor Rer1p. Rer1p binds preferentially immature nicastrin via polar residues within its transmembrane domain that are also critical for interaction with APH-1. Absence of APH-1 significantly increased binding of nicastrin to Rer1p demonstrating the competitive nature of these interactions. Moreover, downregulation of Rer1p promoted while overexpression decreased the formation of full complexes as assessed uby blue-native electrophoresis. Our data indicate that Rer1p expression levels control the formation of -secretase (sub)complexes (and concomitantly total cellular -secretase activity) at a very early stage of complex assembly namely in the formation of the nicastrin-APH-1 subcomplex. Conclusions: We identify Rer1p as a novel limiting factor that negatively regulates -secretase complex assembly during active ERGolgi recycling. This indicates that total cellular -secretase activity is restrained by a secondary ER quality control system that provides a potential therapeutic value.