title: Characterization of APH-1 mutants with a disrupted transmembrane GxxxG motif.
abstract: APH-1 is one of the four essential components of presenilin (PS)-gamma-secretase complexes. There are three major isoforms of APH-1 in humans: APH-1aS, APH-1aL, and APH-1b. To gain insight into the functional role of APH-1 in gamma-secretase complexes, we analyzed the relationship between the three APH-1 forms and characterized APH-1 mutants with a disrupted transmembrane GxxxG motif. We found that overexpression of APH-1aS or APH-1b in human cells significantly reduced the levels of endogenous APH-1aL protein. However, this displacement was not observed in PS-deficient cells, suggesting that it is dependent on PS. In transiently transfected cells, the levels of APH-1aL with G122D or L123D mutations were much lower than wild-type APH-1aL. Also, cycloheximide treatment of stable transfectants revealed that the mutant proteins are much less stable than the wild type. Furthermore, coimmunoprecipitation analysis showed that wild-type but not the mutant APH-1aL is incorporated into PS1 complexes, displacing endogenous APH-1aS. These results collectively indicate that the three forms of APH-1 can replace each other in PS complexes and that the transmembrane GxxxG region is essential for the stability of the APH-1 protein as well as the assembly of PS complexes.
authors.affiliation: Department of Demyelinating Disease and Aging, National Institute of Neuroscience, NCNP, Kodaira, Tokyo 187-8502, Japan. araki@ncnp.go.jp
authors.forename: Wataru
authors.initials: W
authors.lastname: Araki
authors.forename: Shinya
authors.initials: S
authors.lastname: Saito
authors.forename: Noriko
authors.initials: N
authors.lastname: Takahashi-Sasaki
authors.forename: Hirohisa
authors.initials: H
authors.lastname: Shiraishi
authors.forename: Hiroto
authors.initials: H
authors.lastname: Komano
authors.forename: Kiyoko S
authors.initials: KS
authors.lastname: Murayama
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003513:Cycloheximide / Q000378:metabolism; D010450:Endopeptidases; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D010447:Peptide Hydrolases; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017433:Protein Structure, Secondary; D011500:Protein Synthesis Inhibitors / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; D020033:Protein Isoforms; D011500:Protein Synthesis Inhibitors; D003513:Cycloheximide; C475154:APH1A protein, human; D010450:Endopeptidases; D010447:Peptide Hydrolases