Abstract
The presenilin (PS) genes associated with Alzheimer disease encode polytopic transmembrane proteins which undergo physiologic endoproteolytic cleavage to generate stable NH2- and COOH-terminal fragments (NTF or CTF) which co-localize in intracellular membranes, but are tightly regulated in their stoichiometry and abundance. We have used linear glycerol velocity and discontinuous sucrose gradient analysis to investigate the distribution and native conformation of PS1 and PS2 during this regulated processing in cultured cells and in brain. The PS1 NTF and CTF co-localize in the endoplasmic reticulum (ER) and in the Golgi apparatus, where they are components of a approximately 250-kDa complex. This complex also contains beta-catenin but not beta-amyloid precursor protein (APP). In contrast, the PS1 holoprotein precursor is predominantly localized to the rough ER and smooth ER, where it is a component of a approximately 180-kDa native complex. PS2 forms similar but independent complexes. Restricted incorporation of the presenilin NTF and CTF along with a potentially functional ligand (beta-catenin) into a multimeric complex in the ER and Golgi apparatus may provide an explanation for the regulated accumulation of the NTF and CTF.
Highlights
Mutations in the genes encoding the presenilin (PS)1 1 (PS1) and presenilin 2 (PS2) account for the majority of early-onset familial Alzheimer’s disease [1,2,3]
Identification of a Stable 250-kDa Complex of PS1 Endoproteolytic Fragments—To determine the native state of PS1 protein, untransfected HEK293 cells were solubilized in nondenaturing digitonin buffers, and the extracted proteins were fractionated on a linear glycerol velocity gradient
Both NTF and CTF of PS1 were found in identical fractions with a peak in fractions 5 and 6 corresponding to an apparent molecular mass of 250 kDa (Fig. 1A)
Summary
Protein Extraction from Cells—Untransfected HEK293 cells (10-cm dishes) or the transfected HEK293 cell line stably expressing wild type human PS1 and APP695 Cells were washed twice with ice-cold phosphate-buffered saline and resuspended in 10 mM Hepes, pH 7.2, containing protease inhibitors. The homogenate was clarified by centrifugation at 1000 ϫ g for 15 min at 4 °C; the resulting supernatant was centrifuged at 107,000 ϫ g for 1 h at 4 °C, and the pellets were washed once with 10 mM Hepes, pH 7.2, containing protease inhibitors, followed by extraction with lysis buffer as above. Cells (5 ϫ 107) or human brain tissue (0.25 g) were homogenized in 2 ml of sucrose medium (0.25 M sucrose, 5 mM HEPES, 1 mM EDTA, pH 7.2, 5 g/ml chymostatin, pepstatin, leupeptin, and antipain), clarified by centrifugation at 800 ϫ g for 10 min, and fractionated on a discontinuous sucrose gradient. The sedimentation profiles of ER and Golgi membranes in these gradients have been previously characterized [16, 17], specific marker proteins of the ER (calnexin) and Golgi (p58) were detected using monoclonal antibodies (anti-calnexin, Stressgen; anti-p58, Sigma) (12, 19 –21)
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