Evidence that the "NF" motif in transmembrane domain 4 of presenilin 1 is critical for binding with PEN-2.
Macromolecular complexes containing presenilins (PS1 and PS2), nicastrin, anterior pharynx defective phenotype 1 (APH-1), and PS enhancer 2 (PEN-2) mediate the intramembranous, gamma-secretase cleavage of beta-amyloid precursor protein (APP), Notch, and a variety of type 1 membrane proteins. We previously demonstrated that PEN-2 is critical for promoting endoproteolysis of PS1 and that the proximal two-thirds of transmembrane domain (TMD) 1 of PEN-2 is required for binding with PS1. In this study, we sought to identify the structural domains of PS1 that are necessary for binding with PEN-2. To address this issue, we generated a series of constructs encoding PS1 mutants harboring deletions or replacements of specific TMDs of PS1-NTF, and examined the effects of encoded molecules on interactions with PEN-2, stabilization and endoproteolysis of PS1, and gamma-secretase activity. We now show that PS1 TMDs 1 and 2 and the intervening hydrophilic loop are dispensable for binding to PEN-2. Furthermore, analysis of chimeric PS1 molecules that harbor replacements of each TMD with corresponding transmembrane segments from the sterol regulatory element-binding protein cleavage activating protein (SCAP) revealed that the PS1-SCAP TMD4 mutant failed to coimmunoprecipitate endogenous PEN-2, strongly suggesting that the fourth TMD of PS1 is required for interaction with PEN-2. Further mutational analyses revealed that the "NF" sequence within the TMD4 of PS1 is the minimal motif that is required for binding with PEN-2, promoting PS1 endoproteolysis and gamma-secretase activity.
Kim
Seong-Hun
SH
Department of Neurobiology, Pharmacology and Physiology, the University of Chicago, Chicago, Illinois 60637, USA.
Sisodia
Sangram S
SS
D053829:Amyloid Precursor Protein Secretases; D016282:Aspartic Acid Endopeptidases; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D047468:Immunoprecipitation; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D053764:Presenilin-1; D011485:Protein Binding
D008565:Membrane Proteins; C508853:PSEN1 protein, human; C469349:PSENEN protein, human; D053764:Presenilin-1; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human