Presenilin-1 Maintains a Nine-Transmembrane Topology throughout the Secretory Pathway

Dragana Spasic,Alexandra Tolia,Katleen Dillen,Veerle Baert,Bart De Strooper,Stefan Vrijens,Wim Annaert

doi:10.1074/jbc.m600592200

Dragana Spasic, Alexandra Tolia + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m600592200

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Journal: Journal of Biological Chemistry	Publication Date: Sep 1, 2006
Citations: 118	License type: cc-by

Affiliation: KU Leuven

Abstract

Presenilin-1 is a polytopic membrane protein that assembles with nicastrin, PEN-2, and APH-1 into an active gamma-secretase complex required for intramembrane proteolysis of type I transmembrane proteins. Although essential for a correct understanding of structure-function relationships, its exact topology remains an issue of strong controversy. We revisited presenilin-1 topology by inserting glycosylation consensus sequences in human PS1 and expressing the obtained mutants in a presenilin-1 and 2 knock-out background. Based on the glycosylation status of these variants we provide evidence that presenilin-1 traffics through the Golgi after a conformational change induced by complex assembly. Based on our glycosylation variants of presenilin-1 we hypothesize that complex assembly occurs during transport between the endoplasmic reticulum and the Golgi apparatus. Furthermore, our data indicate that presenilin-1 has a nine-transmembrane domain topology with the COOH terminus exposed to the lumen/extracellular surface. This topology is independently underscored by lysine mutagenesis, cell surface biotinylation, and cysteine derivation strategies and is compatible with the different physiological functions assigned to presenilin-1.

Highlights

Of type I membrane proteins such as the amyloid precursor protein (APP) and Notch [4]
This can be explained by the fact that a distance of at least 10 amino acids from any transmembrane domains (TMDs) is required for efficient glycosylation [18], a requirement that is not reached in most human PS1 (hPS1) luminal loop domains
We used a hydrophilic 40-amino acid glycosylation consensus sequence derived from the E. coli leader peptidase that becomes efficiently glycosylated when fused to PS1 fragments [9]

Summary

Introduction

Of type I membrane proteins such as the amyloid precursor protein (APP) and Notch [4]. Western blot revealed that hPS1glyc241 is N-glycosylated but remains endoH sensitive, is partially endoproteolysed (°), and partially rescues NCT maturation, PEN-2 stability, and APP-CTF processing.

Results

Conclusion