Familial Alzheimer's disease-associated mutations block translocation of full-length presenilin 1 to the nuclear envelope.
A polyclonal antibody, M5, to the hydrophilic loop domain of human presenilin 1 (PS1) was prepared. Western blot and immunoprecipitation analyses showed that M5 specifically recognized the processed C-terminal fragment, but not the full-length PS1. Epitope mapping analysis revealed that the essential sequence for recognition of the C-terminal fragment by M5 is DPEAQRR (302-308). The recognition of the C-terminal fragment by M5 in a processing-dependent manner was further confirmed by competitive enzyme-linked immunosorbent assay using the synthetic peptide L281 (281-311), which contains the putative processing site and the preceding amino acids to the site. Although L281 contains the epitope sequence for M5, the maximum inhibition was only 14%. Immunocytochemistry using M5 combined with hL312, which recognizes both full-length PS1 and the C-terminal fragment, allowed us to distinguish the localization of the processed C-terminal fragment from that of full-length PS1. Confocal microscopy demonstrated that the full-length form of wild-type PS1 is preferentially located in the nuclear envelope, while the processed C-terminal fragment is mainly present in the endoplasmic reticulum (ER). However, PS1 with familial Alzheimer's disease-associated mutations could not translocate to the nuclear envelope, and both the full-length and processed mutants were co-localized in the ER.
Honda
T
T
Laboratory for Alzheimer's Disease, Brain Science Institute, RIKEN, 351-0198, Saitama, Japan. tohonda@rc.m-kagaku.co.jp
Nihonmatsu
N
N
Yasutake
K
K
Ohtake
A
A
Sato
K
K
Tanaka
S
S
Murayama
O
O
Murayama
M
M
Takashima
A
A
D000544:Alzheimer Disease / Q000235:genetics*; D000595:Amino Acid Sequence / Q000235:genetics; D001692:Biological Transport; D015153:Blotting, Western; D006801:Humans; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009154:Mutation / Q000502:physiology*; D009685:Nuclear Envelope / Q000378:metabolism* / Q000648:ultrastructure; D010446:Peptide Fragments / Q000378:metabolism; D011233:Precipitin Tests; D053764:Presenilin-1; D014018:Tissue Distribution; D014162:Transfection; D014407:Tumor Cells, Cultured
D008565:Membrane Proteins; C508853:PSEN1 protein, human; D010446:Peptide Fragments; D053764:Presenilin-1