Comparison of Presenilin 1 and Presenilin 2 γ-Secretase Activities Using a Yeast Reconstitution System*
Comparison of Presenilin 1 and Presenilin 2 γ-Secretase Activities Using a Yeast Reconstitution System*
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DOI：
10.1074/jbc.m111.270108
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发表时间：
2011-11
期刊：
影响因子：
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通讯作者：
Y. Yonemura;E. Futai;Sosuke Yagishita;S. Suo;T. Tomita;T. Iwatsubo;S. Ishiura
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文献类型：
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作者：
Y. Yonemura;E. Futai;Sosuke Yagishita;S. Suo;T. Tomita;T. Iwatsubo;S. Ishiura
γ-分泌酶至少由四种蛋白质组成，即早老素(PS)、尼卡斯特素(Nct)、Aph1和Pen2。PS是γ-分泌酶复合体的催化亚基，具有天冬氨酸蛋白酶活性。PS有两个同源基因，即PS1和PS2。为了比较这些含有不同PSS的复合体的活性，我们在缺乏γ-分泌酶同源物的酵母中对它们进行了重组。分别用PS1或PS2、NCT、Pen2、Aph1和人工底物C55-Gal4p转化酵母细胞。底物切割后，Gal4p转位到细胞核，激活报告基因ADE2、HIS3和LacZ的转录。根据酵母菌在选择培养基上的生长情况和γ-半乳糖苷酶活性来测定β-分泌酶活性。在γ-半乳糖苷酶检测中，PS_1的∼活性是PS_2γ-分泌酶的24倍。利用含有γ分泌酶和C55的酵母微粒体，比较了pS1和pS2β-分泌酶产生的Aγ的浓度。PS1γ-分泌酶产生的∼Aβ是PS2γ-分泌酶的24倍。我们发现PS_2产生A-β的最适pH为7.0.与PS_1不同，PS_2复合体含有未成熟的NCT，而PS_1复合体含有成熟的NCT。在这项研究中，我们比较了每一个γ-分泌酶复合体的PS_1或PS_2的活性。利用酵母微粒体共免疫沉淀实验表明，γ-分泌酶复合体中的pS1浓度是pS2的28倍∼。我们的数据表明，在Aβ生产中，PS1复合体的活性仅略低于PS2复合体。
γ-Secretase is composed of at least four proteins, presenilin (PS), nicastrin (NCT), Aph1, and Pen2. PS is the catalytic subunit of the γ-secretase complex, having aspartic protease activity. PS has two homologs, namely, PS1 and PS2. To compare the activity of these complexes containing different PSs, we reconstituted them in yeast, which lacks γ-secretase homologs. Yeast cells were transformed with PS1 or PS2, NCT, Pen2, Aph1, and artificial substrate C55-Gal4p. After substrate cleavage, Gal4p translocates to the nucleus and activates transcription of the reporter genes ADE2, HIS3, and lacZ. γ-Secretase activity was measured based on yeast growth on selective media and β-galactosidase activity. PS1 γ-secretase was ∼24-fold more active than PS2 γ-secretase in the β-galactosidase assay. Using yeast microsomes containing γ-secretase and C55, we compared the concentration of Aβ generated by PS1 or PS2 γ-secretase. PS1 γ-secretase produced ∼24-fold more Aβ than PS2 γ-secretase. We found the optimal pH of Aβ production by PS2 to be 7.0, as for PS1, and that the PS2 complex included immature NCT, unlike the PS1 complex, which included mature NCT. In this study, we compared the activity of PS1 or PS2 per one γ-secretase complex. Co-immunoprecipitation experiments using yeast microsomes showed that PS1 concentrations in the γ-secretase complex were ∼28 times higher than that of PS2. Our data suggest that the PS1 complex is only marginally less active than the PS2 complex in Aβ production.