Pen-2 Is Sequestered in the Endoplasmic Reticulum and Subjected to Ubiquitylation and Proteasome-mediated Degradation in the Absence of Presenilin

Anna Bergman,Emil M Hansson,Sharon E Pursglove,Mark R Farmery,Lars Lannfelt,Urban Lendahl,Johan Lundkvist,Jan Näslund

doi:10.1074/jbc.m313999200

Abstract

The gamma-secretase complex catalyzes intramembrane proteolysis of a number of transmembrane proteins, including amyloid precursor protein, Notch, ErbB4, and E-cadherin. gamma-Secretase is known to contain four major protein constituents: presenilin (PS), nicastrin, Aph-1, and Pen-2, all of which are integral membrane proteins. There is increasing evidence that the formation of the complex and the stability of the individual components are tightly controlled in the cell, assuring correct composition of functional complexes. In this report, we investigate the topology, localization, and mechanism for destabilization of Pen-2 in relation to PS function. We show that PS1 regulates the subcellular localization of Pen-2: in the absence of PS, Pen-2 is sequestered in the endoplasmic reticulum (ER) and not transported to post-ER compartments, where the mature gamma-secretase complexes reside. PS deficiency also leads to destabilization of Pen-2, which is alleviated by proteasome inhibitors. In keeping with this, we show that Pen-2, which adopts a hairpin structure with the N and C termini facing the luminal space, is ubiquitylated prior to degradation and presumably retrotranslocated from the ER to the cytoplasm. Collectively, our data suggest that failure to become incorporated into the gamma-secretase complex leads to degradation of Pen-2 through the ER-associated degradation-proteasome pathway.

Highlights

An increasing number of type 1 membrane proteins have been shown to undergo complex proteolytic processing through a mechanism referred to as regulated intramembrane proteolysis (RIP)1 [1]
We show that PS1 regulates the subcellular localization of Pen-2: in the absence of PS, Pen-2 is sequestered in the endoplasmic reticulum (ER) and not transported to post-ER compartments, where the mature ␥-secretase complexes reside
In this report we demonstrate that PS1 controls the subcellular localization of Pen-2, such that Pen-2 is sequestered in the ER in the absence of PS

Summary

MATERIALS AND METHODS

Antibodies—The polyclonal antibody UD-1 was raised against the N-terminal residues ERVSNEEKLNL of Pen-2. The cells were lysed in IP buffer (25 mM Hepes, pH 7.5, 150 mM KCl, 2 mM EGTA, 1% CHAPS, and protease inhibitor mixture (Roche Applied Science)) and incubated with the indicated primary antibodies over night at 4 °C. Ubiquitylation experiments were conducted with transiently transfected 293T cells and with BD8:Pen-2-HA cells, which were exposed to vehicle only (Me2SO) or MG-132 (5 and 50 ␮M, respectively) for 8 h prior to analysis. CHOPro cells transfected with Pen-2-HA encoding cDNA were exposed to proteinase K (100 ␮g/ml) for 30 min and subsequently analyzed by 10 –20% Tris-Tricine (Invitrogen) SDS-PAGE and immunoblotting. Protein levels were determined by the Bradford method and an equal amount of protein from each cell extract was further analyzed for Pen-2 expression by immunoblotting using the anti-HA antibody.

RESULTS

Findings

DISCUSSION