Membrane topology and nicastrin-enhanced endoproteolysis of APH-1, a component of the gamma-secretase complex.
APH-1, presenilin, nicastrin, and Pen-2 are proteins with varying membrane topologies that compose the gamma-secretase complex, which is responsible for the intramembrane proteolysis of several substrates including the amyloid precursor protein. APH-1 is known to be necessary for gamma-secretase activity, but its precise function in the complex is not fully understood, and its membrane topology has not been described, although it is predicted to traverse the membrane seven times. To investigate this, we used selective permeabilization of the plasma membrane and immunofluorescence microscopy to show that the C terminus of the APH-1 resides in the cytosolic space. Insertion of N-linked glycosylation sites into each of the hydrophilic loop domains and the N terminus of APH-1 showed that the N-terminal domain as well as loops 2, 4, and 6 could be glycosylated, whereas loops 1, 3, and 5 were not. Thus, APH-1 topologically resembles a seven-transmembrane domain receptor with the N terminus and even-numbered loops facing the endoplasmic reticulum lumen, and the C terminus and odd-numbered loops reside in the cytosolic space. By using these glycosylation mutants, we provide evidence that the association between nicastrin and APH-1 may occur very soon after APH-1 synthesis and that the interaction between these two proteins may rely more heavily on the transmembrane domains of APH-1 than on the loop domains. Furthermore, we found that APH-1 can be processed by several endoproteolytic events. One of these cleavages is strongly up-regulated by co-expression of nicastrin and generates a stable C-terminal fragment that associates with nicastrin.
Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
Ryan R
RR
Fortna
Adam S
AS
Crystal
Vanessa A
VA
Morais
Donald S
DS
Pijak
Virginia M-Y
VM
Lee
Robert W
RW
Doms
D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases; D016282:Aspartic Acid Endopeptidases; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D003513:Cycloheximide / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005822:Genetic Vectors; D006031:Glycosylation; D006367:HeLa Cells; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000502:physiology; D008565:Membrane Proteins / Q000378:metabolism / Q000502:physiology*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D010447:Peptide Hydrolases; D017354:Point Mutation; D011233:Precipitin Tests; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D014162:Transfection; D015854:Up-Regulation
D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D011500:Protein Synthesis Inhibitors; C413604:nicastrin protein; D003513:Cycloheximide; C475154:APH1A protein, human; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D010447:Peptide Hydrolases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human