The C-Terminal PAL Motif and Transmembrane Domain 9 of Presenilin 1 Are Involved in the Formation of the Catalytic Pore of the  -Secretase

Abstract

␥-Secretase is an unusual membrane-embedded protease, which cleaves the transmembrane domains (TMDs) of type I membrane proteins, including amyloid-␤ precursor protein and Notch receptor. We have previously shown the existence of a hydrophilic pore formed by TMD6 and TMD7 of presenilin 1 (PS1), the catalytic subunit of ␥-secretase, within the membrane by the substituted cysteine accessibility method. Here we analyzed the structure of TMD8, TMD9, and the C terminus of PS1, which encompass the conserved PAL motif and the hydrophobic C-terminal tip, both being critical for the catalytic activity and the formation of the ␥-secretase complex. We found that the amino acid residues around the PAL motif and the extracellular/luminal portion of TMD9 are highly water accessible and located in proximity to the catalytic pore. Furthermore, the region starting from the luminal end of TMD9 toward the C terminus forms an amphipathic ␣-helix-like structure that extends along the interface between the membrane and the extracellular milieu. Competition analysis using ␥-secretase inhibitors revealed that the TMD9 is involved in the initial binding of substrates, as well as in the subsequent catalytic process as a subsite. Our results provide mechanistic insights into the role of TMD9 in the formation of the catalytic pore and the substrate entry, crucial to the unusual mode of intramembrane proteolysis by ␥-secretase.

Functional Analysis of the Transmembrane Domains of Presenilin 1

“…Plasmid Construction, Cell Culture, Transfection, and Retroviral Infection-cDNAs encoding PS1, APP carrying Swedish mutation (APP NL ), and Notch⌬E were inserted into pMXspuro (9,(11)(12)(13). cDNAs encoding mutant PS1 were generated by long PCR-based QuikChange TM strategy (Stratagene).…”

“…All constructs were sequenced using Thermo Sequenase (USB Corp., Cleveland, OH) on an automated sequencer (Li-Cor, Lincoln, NE) (see Table 1). Maintenance of DKO cells, viral packaging in Plat-E cells, retroviral infection, and generation of stable infectant pools were done as described previously (9,(11)(12)(13)(14).…”

“…Other antibodies were purchased from Chemicon (anti-PS1 loop (MAB5232)), Cell Signaling Technology (anti-cleaved Notch1 (V1744)), Covance (anti-Aph-1aL (O2C2)), Santa Cruz Biotechnology (anti-Nct (N19)), or Sigma (anti-Nct (N1660)). Membrane fractionation, immunoblot analysis, immunoprecipitation of CHAPSO-solubilized lysates, in vitro ␥-secretase assay, cycloheximide treatment, trypsin digestion, or quantitation of A␤ by two-site ELISAs using BNT77 as a capture antibody were performed as described previously (3,8,9,(11)(12)(13)(14)(15)(16)20).…”

“…All methanethiosulfonate (MTS) reagents (Toronto Research Chemicals) were dissolved in dimethyl sulfoxide (DMSO) at 200 mM prior to use or stored at Ϫ80 degree until use. Photoaffinity labeling, SCAM, and cross-linking experiments were performed as described previously (6,9,11,22,23).…”

“…TMD2 and -6 Locate in Proximity to TMD9-We have previously reported the functional role of the extracellular/luminal side of PS1 TMD9 in the initial substrate binding of the ␥-secretase by SCAM using MTS reagents (9,11). Of note, a sulfhydrylbased cross-linking experiment revealed that TMD9 locates in proximity to the residue Leu 250 in TMD6.…”