Membrane topology of gamma-secretase component PEN-2.
PEN-2 is an integral membrane protein that is a necessary component of the gamma-secretase complex, which is central in the pathogenesis of Alzheimer's disease and is also required for Notch signaling. In the absence of PEN-2, Notch signaling fails to guide normal development in Caenorhabditis elegans, and amyloid beta peptide is not generated from the amyloid precursor protein. Human PEN-2 is a 101-amino acid protein containing two putative transmembrane domains. To understand its interaction with other gamma-secretase components, it is important to know the membrane topology of each member of the complex. To characterize the membrane topology of PEN-2, we introduced single amino acid changes in each of the three hydrophilic regions of PEN-2 to generate N-linked glycosylation sites. We found that the N-linked glycosylation sites present in the N- and C-terminal domains of PEN-2 were utilized, whereas a site in the hydrophilic "loop" region connecting the two transmembrane domains was not. The addition of a carbohydrate structure in the N-terminal domain of PEN-2 prevented association with presenilin 1, whereas glycosylation in the C-terminal region of PEN-2 did not, suggesting that the N-terminal domain is important for interactions with presenilin 1. Immunofluorescence microscopy with selective permeabilization of the plasma membrane of cells expressing epitope-tagged forms of PEN-2 confirmed the lumenal location of both the N and C termini. A protease protection assay also demonstrated that the loop domain of PEN-2 is cytosolic. Thus, PEN-2 spans the membrane twice, with the N and C termini facing the lumen of the endoplasmic reticulum.
Crystal
Adam S
AS
Department of Microbiology, The Center for Neurodegenerative Disease Research, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.
Morais
Vanessa A
VA
Pierson
Theodore C
TC
Pijak
Donald S
DS
Carlin
Dan
D
Lee
Virginia M-Y
VM
Doms
Robert W
RW
D053829:Amyloid Precursor Protein Secretases; D016282:Aspartic Acid Endopeptidases; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D010450:Endopeptidases / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006031:Glycosylation; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011233:Precipitin Tests
D017931:DNA Primers; D008565:Membrane Proteins; C469349:PSENEN protein, human; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human