γ-Secretase Exists on the Plasma Membrane as an Intact Complex That Accepts Substrates and Effects Intramembrane Cleavage*

Jay H Chyung,Daniel M Raper,Dennis J Selkoe

doi:10.1074/jbc.m409272200

Jay H Chyung, Daniel M Raper + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m409272200

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Abstract

Research on Alzheimer's disease led to the identification of a novel proteolytic mechanism in all metazoans, the presenilin/gamma-secretase complex. This unique intramembrane-cleaving aspartyl protease is required for the normal processing of Notch, Jagged, beta-amyloid precursor protein (APP), E-cadherin, and many other receptor-like proteins. We recently provided indirect evidence of gamma-secretase activity at the cell surface in HeLa cells following inhibition of receptor-mediated endocytosis. Here, we directly identify and isolate gamma-secretase as an intact complex (Presenilin, Nicastrin, Aph-1, and Pen-2) from the plasma membrane, both in overexpressing cell lines and endogenously. Inhibition of its proteolytic activity allowed cell surface gamma-secretase to be captured in association with its plasma membrane-localized APP substrates (C83 and C99). Moreover, non-denaturing isolation of the intact enzyme complex revealed that cell surface gamma-secretase can specifically generate amyloid beta-protein from an APP substrate and similarly cleave a Notch substrate. These data directly establish the proteolytic function of gamma-secretase on the plasma membrane, independent of a hypothesized substrate trafficking role. We conclude that presenilin/gamma-secretase exists as a mature complex at the cell surface, where it interacts with and can cleave its substrates, consistent with an essential function in processing many adhesion molecules and receptors required for cell-cell interaction or intercellular signaling.

Highlights

Presenilin (PS)1 1 was originally identified as a gene in which missense mutations lead to an aggressive, dominantly inherited form of early-onset Alzheimer’s disease [1]
It is known that PS requires three other integral membrane proteins to effect cleavage within the lipid bilayer [3,4,5], namely Nicastrin (Nct) [6], Aph-1 [7], and Pen-2 [8]. ␥-Secretase cleaves amyloid precursor protein (APP) and numerous other type 1 membrane proteins within their transmembrane domains in a process known as regulated intramembrane proteolysis [9], in which an obligatory first cleavage (e.g. ␤-secretase cleavage of APP in the case of A␤ generation) sheds most of the ectodomain and allows the substrate to undergo subsequent intramembranous cleavage
Direct Biotinylation of Each ␥-Secretase Complex Member at the Cell Surface—First, we attempted to demonstrate the presence of each ␥-secretase complex member on the plasma membrane using surface biotinylation

Summary

Introduction

Presenilin (PS) was originally identified as a gene in which missense mutations lead to an aggressive, dominantly inherited form of early-onset Alzheimer’s disease [1]. Many ␥-secretase substrates are thought to be processed at or near the cell surface, it has been reported that PS resides principally in the endoplasmic reticulum, transGolgi network, and intermediate compartments (18 –20). This apparent discrepancy between PS localization and ␥-secretase activity, referred to as the “spatial paradox” [19, 20], has led some investigators to question whether and how presenilin can mediate ␥-secretase cleavage of plasma membrane receptors. We provide direct evidence for a role of

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