Masking of Transmembrane-Based Retention Signals Controls ER Export of γ-Secretase

Abstract

γ-Secretase is critically involved in the Notch pathway and in Alzheimer's disease. The four subunits of γ-secretase assemble in the endoplasmic reticulum (ER) and unassembled subunits are retained/retrieved to the ER by specific signals. We here describe a novel ERretention/retrieval signal in the transmembrane domain (TMD) 4 of presenilin 1, a subunit of γ-secretase. TMD4 also is essential for complex formation, conferring a dual role for this domain. Likewise, TMD1 of Pen2 is bifunctional as well. It carries an ER-retention/retrieval signal and is important for complex assembly by binding to TMD4. The two TMDs directly interact with each other and mask their respective ER-retention/retrieval signals, allowing surface transport of reporter proteins. Our data suggest a model how assembly of Pen2 into the nascent γ-secretase complex could mask TMD-based ER-retention/retrieval signals to allow plasma membrane transport of fully assembled γ-secretase.

Lack of Evidence for Presenilins as Endoplasmic Reticulum Ca2+ Leak Channels

“…Aspartic acid at position 257 is required for intramolecular cleavage of PS1 into mature fragments. Mutation of this residue causes the accumulation of PS holoprotein in the ER (3,39) (Fig. 3A).…”

Polar Transmembrane-based Amino Acids in Presenilin 1 Are Involved in Endoplasmic Reticulum Localization, Pen2 Protein Binding, and γ-Secretase Complex Stabilization

“…Single Asparagine Mediates Binding of TMD4 PS1 to TMD1 Pen2 -The TMD4 PS1 binds to TMD1 Pen2 (9). To test whether one or more of the polar amino acids in TMD4 PS1 contribute to Pen2 binding, co-immunoprecipitation experiments were performed using transiently transfected CD4-TMD4 PS1 with or without mutations and GFP-tagged Pen2.…”

“…24 h post-transfection, cells were lysed and subjected to immunoprecipitation using GFP antibodies followed by Western blotting with antibodies against ␥-secretase subunits. As a control for a non-complex forming variant PE WNF/AAA was used (7,9), for a mutant forming a complex but being defective in autoproteolysis PE-PALP was used, where the critical PALP motif was deleted (14). PE and PE T197V reconstituted a ␥-secretase complex, indicated by co-precipitation of mature Nct, Aph1, and Pen2 and endoproteolysis to PS1 NTF .…”

“…The subunits nicastrin (Nct) 2 , Pen2, Aph1, and presenilin 1 or 2 (PS) contain one, two, seven, and nine transmembrane domains (TMDs), respectively, which sum up to a total of 19 TMDs in the ␥-secretase complex. Not surprisingly, many important residues were identified in these TMDs, including the catalytically active aspartates in TMD6 and -7 (4), substrate binding sites in TMD2 and -6 (5, 6), protein-protein interaction sites in TMD4 (7)(8)(9), all in PS1. TMD1 of Pen2 contains a binding site for TMD4 PS1 (9), and Aph1 carries residues in TMD3, -4, -5, and -6 that are important for complex formation/stability (10).…”

“…This interaction is dependent on an asparagine in TMD1 (15). In the assembled ␥-secretase, Pen2 interacts with the TMD4 of PS1 (7,8) via this asparagine (9). The TMD4 PS1 also contains an ER retention/retrieval signal, and binding of TMD1 Pen2 to TMD4 PS1 masks the respective signals, allowing export of ␥-secretase out of the ER (9).…”