γ-Secretase cleavage is distinct from endoplasmic reticulum degradation of the transmembrane domain of the amyloid precursor protein

Abstract

One of the critical cleavage events that generates Alzheimer's amyloid Aβ peptide occurs within the transmembrane domain (TMD) of the amyloid precursor protein (APP) and is carried out by a poorly understood enzyme activity known as γ-secretase. To investigate this processing, a probe molecule, H26-57C, was constructed containing the TMD of APP flanked immediately on each side by unique epitope tags. H26-57C-transfected cells secrete a ~2.9-kDa fragment, indicating that the lumenal and cytosolic domains of APP are not required for γ-secretase processing. Pulse-chase experiments indicate that the probe turns over with a half-life of 8 min. No degradation intermediates are detected during the chase period, indicating that TMD turnover is a highly processive mechanism. The protease inhibitors, ALLN and MG132, cause a dramatic (50-fold) increase in the steady-state amount of the probe. All of the inhibitors that prevent degradation of the probe in the rough endoplasmic reticulum increase the amount of the ~2.9- kDa fragment that is secreted into the media and also causes a similar increase the secretion of 4 kDa Aβ from APP-transfected cells. These results indicate that the system responsible for the degradation of the probe in the rough endoplasmic reticulum and the intramembrane cleavage by γ-secretase that produces soluble, secreted Aβ are distinct and opposing processes.