FIGURE 2.

The PS1 L435F mutant displays deficient endoproteolysis but normal cell surface expression. Wild-type (WT) and mutant PS1 were transiently expressed in PS-null MEFs to assess endoproteolytic activity and cell surface localization. Con (A, C) and vector (B) indicate transfection with empty rather than PS1-expressing vector. A, detection of full-length PS1 and PS1-CTF by Western analysis is shown. Proteins were detected with antibodies recognizing the C terminus of PS1 (top panel) and α-tubulin (α-tub, lower panel). Arrows indicate PS1 holoprotein (holo) and CTF. IB, immunoblot. B, quantification of PS1-CTF production is shown. PS1-CTF levels were normalized to α-tubulin and expressed as % of WT levels. Data are the means of three independent experiments (*, p < 0.05 compared with WT PS1-CTF level; n = 3). C, PS1-L435F is delivered to the cell surface and rescues cell-surface localization of mature Nicastrin. PS1-WT and PS1-L435F bearing N-terminal FLAG epitope tags were transiently expressed in PS-null MEFs, and cell surface proteins were isolated by biotinylation and affinity precipitation. PS1and Nicastrin (Nct) were detected by Western analysis of total cell lysates and biotinylated cell surface fractions. The cytosolic protein α-tubulin served as a control for non-cell surface protein. Arrows indicate PS1 holoprotein (holo), PS1 N-terminal fragment (NTF), and immature (imm) and mature (m) forms of Nicastrin.