The topology of pen-2, a γ-secretase subunit, revisited: evidence for a reentrant loop and a single pass transmembrane domain

Abstract

Background: The γ-secretase complex, composed of transmembrane proteins termed presenilin (PS), anterior pharynx defective (APH), nicastrin (NCT), and presenilin enhancer-2 (Pen-2) catalyzes intramembranous hydrolysis of a variety of Type I membrane protein substrates. In order to understand aspects of subunit assembly, interactions, dynamics and catalysis, it is essential to clarify the membrane topology of each polypeptide. Hydophathicity plots predict that the 101 amino acid Pen-2 molecule has two hydrophobic domains (HP1 and HP2) that may serve as transmembrane spanning domains. Earlier reports indicated that transiently overexpressed Pen-2 uses these two hydrophobic domains as transmembrane helices that generates a "U-shaped" hairpin topology with both amino- (N-) and carboxyl-(C-) termini facing the lumen. In this report, we have reexamined the topology of endogenous Pen-2 and Pen-2 chimeras that are stably expressed in mammalian cells, and have assessed the function of these molecules in rescuing γ-secretase activity in Pen-2-deficient fibroblasts.

Results: We confirm that the Pen-2 C-terminus is lumenal, but the N-terminus of Pen-2 is exposed to the cytoplasm, thus indicating that HP1 does not traverse the lipid bilayer as a transmembrane domain. Domain swapping studies reveal the importance of specific regions within the first hydrophobic domain of Pen-2 that are critical for generating the topology that is a prerequisite for mediating PS1 endoproteolysis and γ-secretase activity. Finally, we report that the first fourteen amino acids of the Pen-2 HP1 are required for γ-secretase activity.

Conclusions: We propose that the first hydrophobic domain of Pen-2 forms a structure similar to a reentrant loop while the second hydrophobic domain spans the lipid bilayer.

Fig. 1

The amino-terminus of Pen-2 is in the cytoplasm and the carboxyl-terminus is in the lumen. 10 μg of protein from P15 fractions from HEK 293 cells (Lanes 1–3) or stable N2aWT.11 cells expressing CT11-tagged Pen-2 (Lanes 4–6) were incubated with Proteinase K in the absence or presence of Triton X-100. Resulting reactions were fractionated in 16.5 % Tris/Tricine SDS-PAGE and analyzed by Western blotting with CT11 (a. Lanes 1–6) or PNT-2 (b. Lanes 1–6) antibodies. Molecular mass markers are shown on the left in kilodaltons

Fig. 2

The first hydrophobic region of Pen-2 is not a TMD. a. Sequence of Pen-2 HP1, SREBP TMD2 and Pen-2-SREBP chimeras. b. Cell membranes from stable cell lines expressing CT11 tagged Pen-2 (lanes 1–3) or Pen-2 TMD1 chimera (lanes 4–6) were treated with proteinase K in the absence or presence of Triton X-100 and resulting products were subject to immunoblotting with PNT2 (upper panel) or CT11 (lower panel) antibodies. c. Cell membranes from Pen-2 chimeras with substitutions of segments from the SREBP TMD2 were treated with proteinase K in the absence or presence of Triton X-100 and resulting products were subject to immunoblotting with PNT2 (upper panel) or CT11 (lower panel) antibodies. Molecular mass markers are shown on the left in kilodaltons. d. Pen-2 model proposed in Refs 17, 18 and 19. E. Model for Pen-2 TMD1. F. Revised Pen-2 model

Fig. 3

Rescue of γ-secretase Function in Pen-2-deficient fibroblasts by Pen-2 and the PPS chimera. a. mNΔE Processing in transiently cotransfected cells. b. APPswe processing in transiently cotransfected cells