Mechanism of intramembrane proteolysis investigated with purified rhomboid proteases
Mechanism of intramembrane proteolysis investigated with purified rhomboid proteases
复制标题
DOI：
10.1038/sj.emboj.7600537
复制
发表时间：
2005-02-09
期刊：
影响因子：
11.4
通讯作者：
Freeman, M
中科院分区：
文献类型：
--
作者：
Lemberg, MK;Menendez, J;Freeman, M
膜内蛋白酶具有脂质双层中切割肽键的异常特性，这种环境显然不适合退水的水解反应。这些酶包括位点2蛋白酶，γ-分泌酶/presenilin，信号肽肽酶和菱形，并且具有广泛的细胞功能。所有人都有多个跨膜结构域，并且由于其高疏水性，很难纯化。现在，我们已经开发了一种体外测定，以监测洗涤剂溶解状态下的菱形活性。这使我们能够首次隔离具有催化活性的高度纯菱形。我们的结果表明，洗涤剂 - 溶解的菱形活性模仿了其在许多方面的生物膜活性。对纯化突变蛋白的分析表明，菱形使用丝氨酸蛋白酶催化二元而不是先前提出的三合会。该分析还表明，其他保守的残基参与了配体结合和供水等子公司功能。我们确定菱形和最近发现的德林人之间共享的基因序，该基林参与了错误折叠的膜蛋白的易位。
Intramembrane proteases have the unusual property of cleaving peptide bonds within the lipid bilayer, an environment not obviously suited to a water-requiring hydrolysis reaction. These enzymes include site-2 protease, gamma-secretase/presenilin, signal peptide peptidase and the rhomboids, and they have a wide range of cellular functions. All have multiple transmembrane domains and, because of their high hydrophobicity, have been difficult to purify. We have now developed an in vitro assay to monitor rhomboid activity in the detergent solubilised state. This has allowed us to isolate for the first time a highly pure rhomboid with catalytic activity. Our results suggest that detergent-solubilised rhomboid activity mimics its activity in biological membranes in many aspects. Analysis of purified mutant proteins suggests that rhomboids use a serine protease catalytic dyad instead of the previously proposed triad. This analysis also suggests that other conserved residues participate in subsidiary functions like ligand binding and water supply. We identify a motif shared between rhomboids and the recently discovered derlins, which participate in translocation of misfolded membrane proteins.