Pen-2 Is Essential for γ-Secretase Complex Stability and Trafficking but Partially Dispensable for Endoproteolysis

γ-Secretase is a 19-transmembrane domain (TMD), intramembrane aspartyl protease comprised of presenilin (PS1 or PS2 isoform) as the catalytic component,1 along with nicastrin (Nct), anterior pharynx defective-1 (Aph1αL, Aph1αS, or Aph1β isoform), and presenilin enhancer-2 (Pen-2). All four components are necessary and sufficient for γ-activity.2−4 The γ-secretase complex is responsible for the second and final step in regulated intramembrane proteolysis (RIP)5 of a large and increasing number of substrates, the most studied of which are the β-amyloid precursor protein (APP) and Notch.6 A further complexity of γ-secretase function is the fact that cleavage of the membrane-anchored C99 stub of APP (as a model γ-substrate) occurs at multiple sequential peptide bonds, starting with e-cleavage to release the APP intracellular domain (AICD) from the membrane and leave a 49- or 48-residue amyloid β-protein (Aβ) peptide.7,8 Sequential cleavages of Aβ48/49 every three or four residues moving N-terminally (i.e., every helical turn of the TMD) occur first at the so-called ζ-site to produce Aβ45/46, then at the γ-site to produce Aβ42/43, and finally at the γ′-site to produce Aβ38/40 peptides.9 Aβ42 is generally considered the most pathogenic (i.e., self-aggregating) Aβ species, with an elevated Aβ42/Aβ40 ratio used as a marker of pathogenicity,10 although recently, Aβ43 has also been shown to be pathogenically relevant in vivo.11 The sequential C-terminal trimming of the initial e-cleavage products Aβ48 and Aβ49 by γ-secretase is termed processivity.12 Biochemical studies have previously shown that the Pen-2 subunit is the final component added to γ-secretase,4 whereupon the PS holoprotein undergoes autoproteolysis1,13 in a hydrophobic domain within the cytosolic loop between TMD 6 and 7 to form stable N-terminal and C-terminal fragments (NTF and CTF, respectively) that stay associated in the membrane as heterodimers.14 The essential nature of Pen-2 was illustrated in vivo using Pen-2 knockout mice,15 the embryonic lethal phenotype of which was very similar to that of a PS1/PS2 double knockout or a Notch1 knockout. Pen-2 is very highly conserved, being invariably 101 residues long with 70% identity and 87% similarity among all vertebrates and retaining 56% similarity between Homo sapiens and Arabidopsis thaliana (Figure ​(Figure1a,b).1a,b). Glycosylation studies were utilized to demonstrate that the topology of Pen-2 comprises lumenal N- and C-termini, two TMDs, and a cytosolic loop region.16 The first of these two TMDs was previously reported to be important for interaction with TMD 4 of PS117,18 and was more recently localized to a water-accessible pore.15 The cytosolic loop region of Pen-2 is apparently accessible from the lumenal side of membranes via a hydrophilic cavity and may interact with PS1-CTF.15 Figure 1 Sequence conservation of Pen-2. (a) Multiple-sequence alignment of forms of Pen-2 from various model organisms. The black bar denotes the predicted transmembrane domains. Sequence conservation between Animalia or Vertebrata is shown with asterisks for ... In addition to facilitating PS endoproteolysis, Pen-2 can play other roles. The C-terminal region may be important for γ-secretase complex stability.19 An endoplasmic retention signal sequence in TMD1 may be critical for trafficking.20 Pen-2 is required for γ-secretase complex maturation in a manner independent of PS1 endoproteolysis as shown by a lack of Nct glycosylation in PS1ΔExon9 cells.21 The N-terminal region may affect substrate processivity.22 Adding to the complexity of this protein’s function are two recent reports, one showing that purified PS1 holoprotein can undergo endoproteolysis upon addition of purified Pen-2 alone without a need for Aph1 or Nct23 and the second using Pen-2 knockdown to propose that PS1 can undergo endoproteolysis without Pen-2.24 Because of these multiple putative functions and little knowledge of the mechanisms by which this subunit acts upon γ-secretase, we have used a Pen-2 knockout cell line15 to conduct an extensive mutagenesis screen to investigate Pen-2 cell localization, Pen-2 stability, γ-secretase activity, and γ-secretase maturation.