Localization of presenilin–nicastrin complexes and γ‐secretase activity to the trans‐Golgi network

Abstract Abundant biochemical and genetic evidence suggests that presenilins are catalytic components of γ‐secretase, the protease responsible for generating the Alzheimer amyloid β‐protein. However, the differential localization of presenilins to early secretory compartments and γ‐secretase substrates to late secretory compartments and the plasma membrane (the ‘spatial paradox’) argues against this view. We investigated this issue by studying the localization of nicastrin, another putative γ‐secretase component, and its association with presenilin‐1 into proteolytically active complexes. Glycosidase digests revealed that nicastrin exists in multiple glycoforms and is terminally sialylated, a modification often associated with the trans ‐Golgi network. Trafficking of nicastrin to the trans ‐Golgi network was confirmed by density gradient fractionation and immunofluorescence microscopy. In presenilin‐deficient cells, however, nicastrin trafficking and maturation were abnormal, as the protein was restricted to early secretory compartments and failed to be sialylated. Mature sialylated nicastrin in trans ‐Golgi network fractions was complexed quantitatively with N‐ and C‐terminal fragments of presenilin‐1, whereas immature nicastrin present in early secretory compartments was not. Additionally, trans ‐Golgi network fractions contained the γ‐secretase substrate β‐amyloid precursor protein C83 and were enriched in presenilin‐dependent γ‐secretase proteolytic activity. The results resolve the apparent spatial paradox by demonstrating that presenilin–nicastrin complexes and presenilin‐dependent γ‐secretase activity are co‐localized to a late secretory compartment. The findings provide further evidence that presenilin‐containing complexes are the γ‐secretase, and indicate that presenilins also regulate γ‐secretase assembly.