FIGURE 4.

Amino acids Gly²² and Pro²⁷ of Pen-2 are involved in complex formation and/or stabilization. A, Blue Native PAGE/Western blot analysis of different mutants. Expression of CL Pen-2 in Pen-2^−/− fibroblasts rescues full γ-secretase complex formation (1st lane). In contrast, expression of the G22C mutant compromises complex formation to a large extent (2nd lane). Replacement of the Gly²² with an alanine (G22A) restores complex assembly (3rd lane). Expression of the P27C mutant in Pen-2^−/− fibroblasts results in a partial rescue of γ-secretase complex formation (4th lane). Replacement of the Pro²⁷ with an alanine (P27A) has similar effects as the P27C mutation (5th lane). * indicates the artificial complex composed of PS1 CTF, Aph-1a, and NCT (59). B, SDS-PAGE/Western blot analysis of the Gly²² and Pro²⁷ mutants. The Pen-2 G22C mutant hardly rescues NCT maturation or PS1 endoproteolysis (2nd lane), which is in agreement with the low levels of full complex seen in Blue Native PAGE analysis (A). Replacement of Gly²² by alanine (3rd lane) restores NCT maturation and PS1 endoproteolysis to comparable levels as CL Pen-2 (1st lane). Mutation of the Pro²⁷ to cysteine results in very low Pen-2 levels, probably because of a problem in stability of Pen-2. The Pen-2 P27C and P27A molecules that get assembled in the γ-secretase complex give rise to mature NCT and PS endoproteolysis (4th and 5th lanes). C, cell-free activity assays indicate that the G22C mutant does not rescue AICD production, whereas the G22A mutation rescues AICD production to a large extent. In contrast, the low levels of Pen-2 P27C and Pen-2 P27A that are stably assembled in γ-secretase complexes allow production of equal levels of AICD as the CL Pen-2. AICD levels were corrected for PS1 NTF levels to obtain specific γ-secretase activity. *, p < 0,005.