Mammalian Gup1, a homolog of Saccharomyces cerevisiae glycerol uptake/transporter 1, acts as a negative regulator for N-terminal palmitoylation of Sonic hedgehog
Mammalian Gup1, a homolog of Saccharomyces cerevisiae glycerol uptake/transporter 1, acts as a negative regulator for N-terminal palmitoylation of Sonic hedgehog
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DOI：
10.1111/j.1742-4658.2007.06202.x
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发表时间：
2008-01-01
期刊：
影响因子：
5.4
通讯作者：
Niikura, Takako
中科院分区：
文献类型：
--
作者：
Abe, Yoichiro;Kita, Yoshiko;Niikura, Takako
哺乳动物甘油摄取/转运蛋白 1 (Gup1) 是酿酒酵母 Gup1 的同源物，预计是膜结合 O-酰基转移酶家族的成员，并且与哺乳动物刺猬酰基转移酶（称为 Skn）高度同源，Skn 是果蝇瘦刺猬基因产物的同源物。尽管哺乳动物Gup1在膜结合O-酰基转移酶家族中具有保守的序列，但是该家族的酰基转移酶活性所必需的基序中的组氨酸残基已被亮氨酸取代。在本研究中，我们从成年小鼠肺中克隆了 Gup1 cDNA，并检测了 Gup1 是否参与 Sonic Hedgehog (Shh) N 末端棕榈酰化的调节。使用融合到这些蛋白质每个 C 末端的增强型绿色荧光蛋白的荧光检测到，小鼠 Gup1 的亚细胞定位与小鼠 Skn 的亚细胞定位无法区分。 Gup1 和 Skn 与内质网标记物（78 kDa 葡萄糖调节蛋白）共定位，表明这两个分子与重叠的靶标（包括 Shh）相互作用。事实上，通过使用抗 FLAG IgG 进行免疫沉淀，全长 Shh 与带有 FLAG 标签的 Gup1 共沉淀。使用单克隆抗体 5E1 测定，在缺乏内源性 Skn 的细胞中，具有全长 Shh 的 Gup1 异位表达没有表现出刺猬酰基转移酶活性，单克隆抗体 5E1 被发现在变性条件下识别 Shh 的棕榈酰化 N 端信号结构域。另一方面，Gup1 干扰 COS7 和 NSC34 中内源 Skn 催化的 Shh 棕榈酰化。这些结果表明Gup1是Shh N末端棕榈酰化的负调节因子，可能有助于Shh的多种生物学作用。
Mammalian glycerol uptake/transporter 1 (Gup1), a homolog of Saccharomyces cerevisiae Gup1, is predicted to be a member of the membrane-bound O-acyltransferase family and is highly homologous to mammalian hedgehog acyltransferase, known as Skn, the homolog of the Drosophila skinny hedgehog gene product. Although mammalian Gup1 has a sequence conserved among the membrane-bound O-acyltransferase family, the histidine residue in the motif that is indispensable to the acyltransferase activity of the family has been replaced with leucine. In this study, we cloned Gup1 cDNA from adult mouse lung and examined whether Gup1 is involved in the regulation of N-terminal palmitoylation of Sonic hedgehog (Shh). Subcellular localization of mouse Gup1 was indistinguishable from that of mouse Skn detected using the fluorescence of enhanced green fluorescent protein that was fused to each C terminus of these proteins. Gup1 and Skn were co-localized with an endoplasmic reticulum marker, 78 kDa glucose-regulated protein, suggesting that these two molecules interact with overlapped targets, including Shh. In fact, full-length Shh coprecipitated with FLAG-tagged Gup1 by immunoprecipitation using anti-FLAG IgG. Ectopic expression of Gup1 with full-length Shh in cells lacking endogenous Skn showed no hedgehog acyltransferase activity as determined using the monoclonal antibody 5E1, which was found to recognize the palmitoylated N-terminal signaling domain of Shh under denaturing conditions. On the other hand, Gup1 interfered with the palmitoylation of Shh catalyzed by endogenous Skn in COS7 and NSC34. These results suggest that Gup1 is a negative regulator of N-terminal palmitoylation of Shh and may contribute to the variety of biological actions of Shh.