Presenilin-1 (PS1) facilitates ␥-secretase cleavage of the ␤-amyloid precursor protein and the intramembraneous cleavage of Notch1. Although Alzheimer's diseaseassociated mutations in the homologous presenilin (PS2) gene elevate amyloid ␤-peptide (A␤42) production like PS1 mutations, here we demonstrate that a gene ablation of PS2 (unlike that of PS1) in mice does not result in a severe phenotype resembling that of Notchablated animals. To investigate the amyloidogenic function of PS2 more directly, we mutagenized a conserved aspartate at position 366 to alanine, because the corresponding residue of PS1 is known to be required for its amyloidogenic function. Cells expressing the PS2 D366A mutation exhibit significant deficits in proteolytic processing of ␤-amyloid precursor protein indicating a defect in ␥-secretase activity. The reduced ␥-secretase activity results in the almost complete inhibition of A␤ and p3 production in cells stably expressing PS2 D366A, whereas cells overexpressing the wild-type PS2 cDNA produce robust levels of A␤ and p3. Using highly sensitive in vivo assays, we demonstrate that the PS2 D366A mutation not only blocks ␥-secretase activity but also inactivates PS2 activity in Notch signaling by inhibiting the proteolytic release of the cytoplasmic Notch1 domain. These data suggest that PS2 is functionally involved in A␤ production and Notch signaling by facilitating similar proteolytic cleavages.

The two homologous presenilins are key factors for the generation of amyloid ␤-peptide (A␤), since Alzheimer's disease (AD)-associated mutations enhance the production of the pathologically relevant 42-amino acid A␤ (A␤42), and a gene knockout of presenilin-1 (PS1) significantly inhibits total A␤ production. Presenilins undergo proteolytic processing within the domain encoded by exon 9, a process that may be closely related to their biological and pathological activity. An AD-associated mutation within the PS1 gene deletes exon 9 (PS1⌬exon9) due to a splicing error and results in the accumulation of the uncleaved full-length protein. We now demonstrate the unexpected finding that the pathological activity of PS1⌬exon9 is independent of its lack to undergo proteolytic processing, but is rather due to a point mutation (S290C) occurring at the aberrant exon 8/10 splice junction. Mutagenizing the cysteine residue at position 290 to the original serine residue completely inhibits the pathological activity in regard to the elevated production of A␤42. Like PS1⌬exon9, the resulting presenilin variant (PS1⌬exon9 C290S) accumulates as an uncleaved protein and fully replaces endogenous presenilin fragments. Moreover, PS1⌬exon9 C290S exhibits a significantly increased biological activity in a highly sensitive in vivo assay as compared with the ADassociated mutation. Therefore not only the increased A␤42 production but also the decreased biological function of PS1⌬exon9 is due to a point mutation and independent of the lack of proteolytic processing.

Mutations in the presenilin-1 (PS1) gene are associated with Alzheimer's disease and cause increased secretion of the neurotoxic amyloid-beta peptide (Abeta). Critical intramembraneous aspartates at residues 257 and 385 are required for the function of PS1 protein. Here we investigate the biological function of a naturally occurring PS1 splice variant (PS1 Deltaexon 8), which lacks the critical aspartate 257. Cell lines that stably express PS1 Deltaexon 8 or a PS1 protein in which aspartate residue 257 is mutated secrete significant levels of Abeta, whereas Abeta generation is severely reduced in cells transfected with PS1 containing a mutation of aspartate 385. In contrast, endoproteolytic processing of Notch is almost completely inhibited in cell lines expressing any of the PS1 variants that lack one of the critical aspartates. These data indicate that PS1 may differentially facilitate gamma-secretase-mediated generation of Abeta and endoproteolysis of Notch.

The first membrane-spanning domain (m1) of the model cis Golgi protein M (formerly called E1) from the avian coronavirus infectious bronchitis virus is required for targeting to the Golgi complex. When inserted in place of the membrane-spanning domain of a plasma membrane protein (vesicular stomatitis virus G protein), the chimeric protein ("Gm1") is retained in the Golgi complex of transfected cells. To determine the precise features of the m1 domain responsible for Golgi targeting, we produced single amino acid substitutions in m1 and analyzed their effects on localization of Gm1. Expression at the plasma membrane was used as the criterion for loss of Golgi retention. Rates of oligosaccharide processing were used as a measure of rate and efficiency of transport through the Golgi complex. We identified four uncharged polar residues that are critical for Golgi retention of Gm1 (Asn465, Thr469, Thr476, and Gln480). These residues line one face of a predicted alpha-helix. Interestingly, when the m1 domain of the homologous M protein from mouse hepatitis virus is inserted into the G protein reporter, the chimeric protein is not efficiently retained in the Golgi complex, but transported to the cell surface. Although it possesses three of the four residues we identified as important in the avian m1 sequence, other residues in the membrane-spanning domain from the mouse protein must prevent efficient recognition of the polar face within the lipid bilayer of the cis Golgi.