Membrane targeting of L-type calcium channels -: Role of palmitoylation in the subcellular localization of the β2a subunit
Membrane targeting of L-type calcium channels -: Role of palmitoylation in the subcellular localization of the β2a subunit
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DOI：
10.1074/jbc.273.36.23590
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发表时间：
1998-09-04
影响因子：
4.8
通讯作者：
Hosey, MM
中科院分区：
文献类型：
--
作者：
Chien, AJ;Gao, TY;Hosey, MM
在这项研究中，我们报告了棕榈酰化是大鼠电压依赖性钙通道β (2a)亚基亚细胞定位的关键决定因素。转染细胞的免疫组织化学染色显示，棕榈酰化缺陷β (2a)亚基呈现弥漫的细胞内染色模式，与野生型β (2a)亚基的质膜分布形成鲜明对比。出乎意料的是，Cys(3)和Cys(4)棕榈酰化位点远端区域的突变影响了β (2a)蛋白的棕榈酰化。β (2a)亚基src同源3基序的突变影响β (2a)蛋白的棕榈酰化和亚细胞定位，β相互作用域的突变破坏了表达的α(1)和β亚基之间的相互作用，也导致β (2a)蛋白的棕榈酰化和弥漫细胞内定位降低。嵌合蛋白的研究表明，β (2a)亚基的16个氨基酸N端足以将棕榈酰化赋予非棕榈酰化的β (1b)和β(3)亚型。然而，嵌合β亚基的棕榈酰化本身不足以恢复野生型β (2a)蛋白所观察到的质膜定位。用brefeldin A处理转染的细胞增加了β (2a)蛋白中棕榈酸的掺入量，表明β (2a)的棕榈酰化发生在蛋白质合成期间或之后不久。另外两种β(2)变体，兔β (2a)和β (2b)，在Cys(3)和Cys(4)缺乏棕榈酰化，表现出弥漫性细胞内染色模式，没有棕榈酰化。
In this study, we report that palmitoylation was a critical determinant of the subcellular localization of the rat beta(2a) subunit of voltage-dependent calcium channels. Immunohistochemical staining of transfected cells revealed that a palmitoylation-deficient beta(2a) subunit exhibited a diffuse intracellular staining pattern, in contrast to the plasma membrane distribution seen with the wild-type beta(2a) subunit. Unexpectedly, mutations in regions distal to the palmitoylation sites at Cys(3) and Cys(4) affected palmitoylation of the beta(2a) protein. Mutations in an src homology 3 motif of the beta(2a) subunit affected both palmitoylation and subcellular localization of the beta(2a) protein, A mutation in the beta interaction domain, which disrupted interactions between the expressed alpha(1) and beta subunits, also resulted in a decreased palmitoylation and diffuse intracellular localization of the beta(2a) protein. Studies of chimeric proteins revealed that the 16-amino acid N terminus of the beta(2a) subunit was sufficient to confer palmitoylation to the nonpalmitoylated beta(1b) and beta(3) isoforms. However, palmitoylation of chimeric beta subunits was by itself insufficient to restore the plasma membrane localization observed with the wild-type beta(2a) protein. Treatment of transfected cells with brefeldin A increased the amount of palmitic acid incorporated in the beta(2a) protein, suggesting that palmitoylation of beta(2a) occurs during or shortly after protein synthesis. Two other beta(2) variants, the rabbit beta(2a) and beta(2b), which lack the palmitoylation sties at Cys(3) and Cys(4), exhibited a diffuse intracellular staining pattern and were not palmitoylated.