Phosphorylation, subcellular localization, and membrane orientation of the Alzheimer's disease-associated presenilins.
Presenilins 1 and 2 are unglycosylated proteins with apparent molecular mass of 45 and 50 kDa, respectively, in transfected COS-1 and Chinese hamster ovary cells. They colocalize with proteins from the endoplasmic reticulum and the Golgi apparatus in transfected and untransfected cells. In COS-1 cells low amounts of intact endogeneous presenilin 1 migrating at 45 kDa are detected together with relative larger amounts of presenilin 1 fragments migrating between 18 and 30 kDa. The presenilins have a strong tendency to form aggregates (mass of 100-250 kDa) in SDS-polyacrylamide gel electrophoresis, which can be partially resolved when denatured by SDS at 37 degrees C instead of 95 degrees C. Sulfation, glycosaminoglycan modification, or acylation of the presenilins was not observed, but both proteins are posttranslationally phosphorylated on serine residues. The mutations Ala-246 --> Glu or Cys-410 --> Tyr that cause Alzheimer's disease do not interfere with the biosynthesis or phosphorylation of presenilin 1. Finally, using low concentrations of digitonin to selectively permeabilize the cell membrane but not the endoplasmic reticulum membrane, it is demonstrated that the two major hydrophilic domains of presenilin 1 are oriented to the cytoplasm. The current investigation documents the posttranslational modifications and subcellular localization of the presenilins and indicates that postulated interactions with amyloid precursor protein metabolism should occur in the early compartments of the biosynthetic pathway.
De Strooper
B
B
Experimental Genetics Group, Belgium. bart.destrooper@med.kuleuven.ac.be
Beullens
M
M
Contreras
B
B
Levesque
L
L
Craessaerts
K
K
Cordell
B
B
Moechars
D
D
Bollen
M
M
Fraser
P
P
George-Hyslop
P S
PS
Van Leuven
F
F
D000544:Alzheimer Disease / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002462:Cell Membrane / Q000737:chemistry; D006224:Cricetinae; D019084:Fluorescent Antibody Technique, Indirect; D008565:Membrane Proteins / Q000737:chemistry*; D008958:Models, Molecular; D010766:Phosphorylation; D053764:Presenilin-1; D053766:Presenilin-2; D011499:Protein Processing, Post-Translational; D013347:Subcellular Fractions / Q000737:chemistry*; D014162:Transfection
D008565:Membrane Proteins; D053764:Presenilin-1; D053766:Presenilin-2