Specific domains in anterior pharynx-defective 1 determine its intramembrane interactions with nicastrin and presenilin

Abstract

γ-Secretase, a multisubunit transmembrane protease comprised of presenilin, nicastrin, presenilin enhancer 2, and anterior pharynx-defective one, participates in the regulated intramembrane proteolysis of Type I membrane proteins including the amyloid precursor protein (APP). Although Aph-1 is thought to play a structural role in the assembly of γ-secretase complex and several transmembrane domains (TMDs) of Aph-1 have been shown to be critical for its function, the importance of the other domains of Aph-1 remains elusive. We screened a series of Aph-1 mutants and focused on nine mutations distributed in six different TMDs of human APH-1aS, assessing their ability to complement mouse embryonic fibroblasts lacking Aph-1. We showed that mutations in TMD4 (G126) and TMD5 (H171) of Aph-1aS prevented the formation of the Nct/Aph-1 subcomplex. Importantly, although mutations in TMD3 (Q83/E84/R85) and TMD6 (H197) of APH-1aS did not affect Nct/Aph-1 subcomplex formation, both mutations prevented further association/endoproteolysis of PS1. We propose a model that identifies critical TMDs of Aph-1 for associations with Nct and PS for the stepwise assembly of γ-secretase components.

Fig. 1

Topology of Aph-1 depicted by Residue-based Diagram Editor (Campagne and Weinstein, 1999;Konvicka, et al., 2000;Skrabanek, et al., 2003). The residues in TMDs targeted for mutation highlighted in gray.

Figs. 2A-F

Protein blot analysis of various hAPH-1aS mutations in complementing structure and function of γ-secretase. Immortalized Aph-1 deficient cells transiently transfected with expression plasmids encoding mutant hAPH-1aS and human APP695. Cell extracts were prepared and subjected to protein blot analysis using antisera specific for: A. Nct; glycosylated (arrowhead) and nonglycosylated (arrow) Nct; B. PS1-CTF (processed C-terminal fragment of PS1, arrowhead); C. V5-tagged hAPH-1aS; and D. Actin. E. Quantification of γ-secretase maturation by Nct glycosylation (panel A, arrowhead; mean±SEM). F. Assessment of APP processing as determined by secretion Aβ-40 (mean±SEM). Conditioned culture media were collected after transfection and analyzed by sandwich ELISA. * indicates p<0.05.

Figs. 2A-F

Protein blot analysis of various hAPH-1aS mutations in complementing structure and function of γ-secretase. Immortalized Aph-1 deficient cells transiently transfected with expression plasmids encoding mutant hAPH-1aS and human APP695. Cell extracts were prepared and subjected to protein blot analysis using antisera specific for: A. Nct; glycosylated (arrowhead) and nonglycosylated (arrow) Nct; B. PS1-CTF (processed C-terminal fragment of PS1, arrowhead); C. V5-tagged hAPH-1aS; and D. Actin. E. Quantification of γ-secretase maturation by Nct glycosylation (panel A, arrowhead; mean±SEM). F. Assessment of APP processing as determined by secretion Aβ-40 (mean±SEM). Conditioned culture media were collected after transfection and analyzed by sandwich ELISA. * indicates p<0.05.

Figs. 3A-F

BN-PAGE analysis of selected mutants on the formation of γ-secretase holocomplex and Nct/Aph-1 subcomplex. Membrane fractions of Aph-1 deficient cells transfected with mutant hAPH-1aS were prepared and subjected to blue native gel analysis using antiserum recognizing PS1-CTF (A), Pen-2 (B), Nct (C), and transfected hAph-1 (D). Note that PS and Pen-2 staining showed only holocomplex whereas Nct staining revealed both γ-secretase holocomplex and Nct/Aph-1 subcomplex. V5 staining showed the corresponding holocomplex and subcomplex, albeit with high background due to overexpression of hAPH-1. Hollow arrowhead, holocomplex; arrow, Nct/Aph-1 subcomplex; filled arrowhead, non-specific band as observed also in loading control; *, faint γ-secretase holocomplex by H171 complementation. E and F. Quantification (mean±SEM) of γ-secretase holocomplex (in panel E) and Nct/Aph-1 subcomplex (in panel F) using band intensity of the Nct blot (panel C). * indicates p<0.05.

Figs. 3A-F

BN-PAGE analysis of selected mutants on the formation of γ-secretase holocomplex and Nct/Aph-1 subcomplex. Membrane fractions of Aph-1 deficient cells transfected with mutant hAPH-1aS were prepared and subjected to blue native gel analysis using antiserum recognizing PS1-CTF (A), Pen-2 (B), Nct (C), and transfected hAph-1 (D). Note that PS and Pen-2 staining showed only holocomplex whereas Nct staining revealed both γ-secretase holocomplex and Nct/Aph-1 subcomplex. V5 staining showed the corresponding holocomplex and subcomplex, albeit with high background due to overexpression of hAPH-1. Hollow arrowhead, holocomplex; arrow, Nct/Aph-1 subcomplex; filled arrowhead, non-specific band as observed also in loading control; *, faint γ-secretase holocomplex by H171 complementation. E and F. Quantification (mean±SEM) of γ-secretase holocomplex (in panel E) and Nct/Aph-1 subcomplex (in panel F) using band intensity of the Nct blot (panel C). * indicates p<0.05.

Fig. 4A-G

Co-IP analysis using WT or mutant 6his-tagged hAPH-1aS. Aph-1 deficient cells transfected with complex assembly defective Aph-1aS mutants were prepared as for analysis by BN-PAGE. Lysates, subjected to immunoprecipation and SDS- PAGE, were immunoblotted for: A. PS1-CTF (arrow); B and C. Nct; Shorter (B) and longer (C) exposure of Nct blot. Arrowhead, glycosylated Nct; arrow, nonglycosylated Nct; D. hAPH1aS (arrow); and E. Actin. T, total lysate; P, pulled-down fraction. F. Aph-1 mutations differentially disrupt steps of γ-secretase maturation. G. Proposed model of domain-specific functions of Aph-1 in interaction with Nct and Ps/Pen-2.

Fig. 4A-G

Co-IP analysis using WT or mutant 6his-tagged hAPH-1aS. Aph-1 deficient cells transfected with complex assembly defective Aph-1aS mutants were prepared as for analysis by BN-PAGE. Lysates, subjected to immunoprecipation and SDS- PAGE, were immunoblotted for: A. PS1-CTF (arrow); B and C. Nct; Shorter (B) and longer (C) exposure of Nct blot. Arrowhead, glycosylated Nct; arrow, nonglycosylated Nct; D. hAPH1aS (arrow); and E. Actin. T, total lysate; P, pulled-down fraction. F. Aph-1 mutations differentially disrupt steps of γ-secretase maturation. G. Proposed model of domain-specific functions of Aph-1 in interaction with Nct and Ps/Pen-2.

Fig. 4A-G

Co-IP analysis using WT or mutant 6his-tagged hAPH-1aS. Aph-1 deficient cells transfected with complex assembly defective Aph-1aS mutants were prepared as for analysis by BN-PAGE. Lysates, subjected to immunoprecipation and SDS- PAGE, were immunoblotted for: A. PS1-CTF (arrow); B and C. Nct; Shorter (B) and longer (C) exposure of Nct blot. Arrowhead, glycosylated Nct; arrow, nonglycosylated Nct; D. hAPH1aS (arrow); and E. Actin. T, total lysate; P, pulled-down fraction. F. Aph-1 mutations differentially disrupt steps of γ-secretase maturation. G. Proposed model of domain-specific functions of Aph-1 in interaction with Nct and Ps/Pen-2.