Alzheimer’s disease (AD) is the most frequent form of dementia found in the elderly. The Aβ peptides that are the principal component of the amyloid plaques in the brains of AD patients are released to the extracellular space by the activity of g-secretase. The g-secretase is a multimeric, high-molecular-weight complex composed of presenilin (PS), nicastrin, anterior pharynx-defective phenotype 1 (Aph1) and PS enhancer 2 (Pen2). In previous work a quality control mechanism for g-secretase assembly was suggested that is based on retention signals, which keep unassembled subunits and assembly intermediates in the endoplasmic reticulum (ER). These signals become inactivated in the properly assembled complex and the g-secretase is transported to the plasma membrane and the endosomes where it is active. It was the aim of the present study to provide evidence for the proposed quality control hypothesis for g-secretase assembly.
The first step in this work was the identification of ER retention signals in the different subunits of g-secretase. By applying a reporter protein approach, two novel ER retention signals were identified in the transmembrane domain (TMD) 4 of PS1 and the TMD1 of Pen2. Both signals are based on polar residues in the hydrophobic context of a TMD. The signal in the Pen2-TMD1 also contributes to the stability of Pen2. Mutagenesis of the two known signals in PS1 (TMD4 and TMD9) did not result in the ER export of full-length PS1. Thus, it is suggested here that further signals exist in PS1, either in the remaining 7 TMDs or in the cytoplasmic domains.
The second part of the present study aimed on the identification of the machinery accomplishing TMD-based ER retention. Retention in the ER 1 (Rer1) was identified as a protein involved in the ER retention of Pen2 but not PS1. Pen2 was the first identified mammalian substrate of Rer1 and Rer1 interacts with Pen2 via the binding to the Pen2-TMD1. The signal in the PS1-TMD4 is independent of Rer1, suggesting that other, so far unknown mechanisms exist which contribute to TMD-based ER retention.
Third, the mechanisms by which ER retention signals become inactivated in the assembled g-secretase were elucidated. Data from our lab and by others already demonstrated that the Pen2-TMD1 and the PS1-TMD4 are necessary for the interaction of PS1 and Pen2. Thus, both TMDs are bifunctional, since they contribute to protein-protein interaction and to ER retention. It was shown here, that the protein-protein interaction between the TMDs results in the masking and inactivation of the retention signals in both TMDs. In this way both functions of the TMDs are connected and this mechanism can be used by the ER quality control to probe the assembly status of g-secretase. Most likely, this mechanism can be generalised for other multimeric proteins like ion channels and cell surface receptors.
Furthermore, it was shown that the stable overexpression of the PS1-TMD4 interferes with the described quality control mechanism and thus with the assembly of the g-secretase. This finding highlights the relevance of TMDs for g-secretase function and it discloses the possibility that pharmacological targeting of g-secretase TMDs could be used to inhibit this important enzyme under pathological conditions.