Analysis of DHHC Acyltransferases Implies Overlapping Substrate Specificity and a Two-Step Reaction Mechanism
Analysis of DHHC Acyltransferases Implies Overlapping Substrate Specificity and a Two-Step Reaction Mechanism
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DOI：
10.1111/j.1600-0854.2009.00925.x
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发表时间：
2009-08-01
期刊：
影响因子：
4.5
通讯作者：
Ungermann, Christian
中科院分区：
文献类型：
--
作者：
Hou, Haitong;Peter, Arun T. John;Ungermann, Christian
Asp-His-His-Cys（DHHC）富含半胱氨酸结构域（CRD）酰基转移酶是多位跨膜蛋白，其沿着真核细胞的内膜系统发现，并介导外周和完整膜蛋白的棕榈酰化。在这里，我们解决了在体内底物特异性的7 DHHC酰基转移酶外周膜蛋白的过表达的方法。对于所有分析的DHHC蛋白，我们检测到强烈重叠的底物特异性。此外，我们现在显示了Pfa 5的酰基转移酶活性。更重要的是，DHHC蛋白Pfa 3能够在液泡中捕获几种底物。对于Pfa 3及其底物Vac 8，我们可以区分酰化反应中的两个连续步骤：初始结合独立于DHHC盒中的中央半胱氨酸发生，但需要其底物Vac 8的豆蔻酰化，以及DHHC基序依赖性酰化。我们的数据还表明，蛋白质可以棕榈酰化的几个细胞器。因此，DHHC蛋白的细胞内分布提供了酰基转移酶网络，其可以促进底物蛋白的动态膜缔合。
Asp-His-His-Cys (DHHC) cysteine-rich domain (CRD) acyltransferases are polytopic transmembrane proteins that are found along the endomembrane system of eukaryotic cells and mediate palmitoylation of peripheral and integral membrane proteins. Here, we address the in vivo substrate specificity of five of the seven DHHC acyltransferases for peripheral membrane proteins by an overexpression approach. For all analysed DHHC proteins we detect strongly overlapping substrate specificity. In addition, we now show acyltransferase activity for Pfa5. More importantly, the DHHC protein Pfa3 is able to trap several substrates at the vacuole. For Pfa3 and its substrate Vac8, we can distinguish two consecutive steps in the acylation reaction: an initial binding that occurs independently of its central cysteine in the DHHC box, but requires myristoylation of its substrate Vac8, and a DHHC-motif dependent acylation. Our data also suggest that proteins can be palmitoylated on several organelles. Thus, the intracellular distribution of DHHC proteins provides an acyltransferase network, which may promote dynamic membrane association of substrate proteins.