MEMBRANE INSERTION AND ASSEMBLY OF DUCTIN - A POLYTOPIC CHANNEL WITH DUAL ORIENTATIONS
MEMBRANE INSERTION AND ASSEMBLY OF DUCTIN - A POLYTOPIC CHANNEL WITH DUAL ORIENTATIONS
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DOI：
10.1002/j.1460-2075.1995.tb00030.x
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发表时间：
1995-08-01
期刊：
影响因子：
11.4
通讯作者：
FINBOW, ME
中科院分区：
文献类型：
--
作者：
DUNLOP, J;JONES, PC;FINBOW, ME
Ductin是一种高度保守的多聚跨膜蛋白，是液泡H+- atp酶（v - atp酶）的亚基c组分，也是间隙连接的连接子通道的组分。先前的研究表明，v - atp酶中的导管蛋白与连接子中的导管蛋白取向相反。利用一个体外翻译系统，结合来自内质网的微粒体，我们发现ductin被共翻译插入到膜双分子层中，这表明ductin的合成依赖于信号识别颗粒。通过连接β -内酰胺酶衍生的c端多肽和使用半胱氨酸替代偶联化学标记，我们发现ductin以相似的比例以两个方向插入微粒体膜。相比之下，鱿鱼视紫红质似乎以单一方向插入。改变导管蛋白n端的守恒带电残基不影响两个取向的比例。一旦进入微粒体膜，导管蛋白就会组装成一个低聚复合物，其中包含一个可被水溶性探针访问的孔，这让人想起在v - atp酶和连接子中发现的导管蛋白复合物。
Ductin is a highly conserved and polytopic transmembrane protein which is the subunit c component of the vacuolar H+-ATPase (V-ATPase) and a component of a connexon channel of gap junctions. Previous studies have suggested that ductin in the V-ATPase has the opposite orientation of ductin in a connexon. Using an in vitro translation system coupled to microsomes derived from the endoplasmic reticulum, we show that ductin is co-translationally inserted into the membrane bilayer, suggesting a dependency on the signal recognition particle for synthesis. By attaching a C-terminal polypeptide derived from beta-lactamase and by using cysteine replacement coupled to chemical labelling, we show that ductin is inserted into the microsomal membrane in both orientations in similar proportions. In contrast, squid rhodopsin appears to be inserted in a single orientation. Changing conserved charged residues at the N-terminus of ductin does not affect the ratio of the two orientations. Once in the microsomal membrane, ductin assembles into an oligomeric complex which contains a pore accessible to a water-soluble probe, reminiscent of the ductin complex found in the V-ATPase and a connexon.