Ste24p Mediates Proteolysis of Both Isoprenylated and Non-prenylated Oligopeptides
Ste24p Mediates Proteolysis of Both Isoprenylated and Non-prenylated Oligopeptides
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DOI：
10.1074/jbc.m116.718197
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发表时间：
2016-07-01
影响因子：
4.8
通讯作者：
Schmidt, Walter K.
中科院分区：
文献类型：
--
作者：
Hildebrandt, Emily R.;Arachea, Buenafe T.;Schmidt, Walter K.
Rce1p 和 Ste24p 是参与异戊二烯化蛋白的蛋白水解成熟的整合膜蛋白。大量已发表的证据表明，Rce1p 需要异戊二烯基部分作为重要的底物决定因素。相比之下，我们报告 Ste24p 可以在体外裂解异戊二烯化和非异戊二烯化底物，这表明异戊二烯基部分不是底物识别所必需的。异戊二烯化底物与非异戊二烯化底物的稳态酶动力学显着不同，强烈暗示底物-膜相互作用在蛋白酶功能中的作用。质谱分析确定了异戊二烯化和非异戊二烯化底物中 P1 为脂肪族的键的裂解偏好，尽管这不一定是预测性的。所识别的切割位点相对于 C 末端不处于固定距离位置。在本研究中，Ste24p 裂解的底物基于已知的异戊二烯化蛋白质（即 K-Ras4b 和酵母 a 因子交配信息素）和非异戊二烯化生物肽（A 链和胰岛素链），它们是可溶性锌依赖性金属蛋白酶 M16A 家族的已知底物。这些结果表明，Ste24p 的底物谱比预期更广泛，与 M16A 蛋白酶家族的底物谱比与其功能相关的 Rce1p CAAX 蛋白酶更相似。
Rce1p and Ste24p are integral membrane proteins involved in the proteolytic maturation of isoprenylated proteins. Extensive published evidence indicates that Rce1p requires the isoprenyl moiety as an important substrate determinant. By contrast, we report that Ste24p can cleave both isoprenylated and non-prenylated substrates in vitro, indicating that the isoprenyl moiety is not required for substrate recognition. Steady-state enzyme kinetics are significantly different for prenylated versus non-prenylated substrates, strongly suggestive of a role for substrate-membrane interaction in protease function. Mass spectroscopy analyses identify a cleavage preference at bonds where P1 is aliphatic in both isoprenylated and non-prenylated substrates, although this is not necessarily predictive. The identified cleavage sites are not at a fixed distance position relative to the C terminus. In this study, the substrates cleaved by Ste24p are based on known isoprenylated proteins (i.e. K-Ras4b and the yeast a-factor mating pheromone) and non-prenylated biological peptides (A and insulin chains) that are known substrates of the M16A family of soluble zinc-dependent metalloproteases. These results establish that the substrate profile of Ste24p is broader than anticipated, being more similar to that of the M16A protease family than that of the Rce1p CAAX protease with which it has been functionally associated.