180-kD ribosome receptor is essential for both ribosome binding and protein translocation.
180-kD ribosome receptor is essential for both ribosome binding and protein translocation.
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DOI：
10.1083/jcb.120.4.853
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发表时间：
1993-02
期刊：
影响因子：
--
通讯作者：
Meyer DI
中科院分区：
文献类型：
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作者：
Savitz AJ;Meyer DI
我们以前已经分离出一个180 kD的核糖体受体（p180）从哺乳动物粗糙的ER，当纳入脂质体，结合核糖体的亲和力类似于完整的膜。为了直接评估p180对核糖体结合以及蛋白质易位的贡献，使用单克隆抗体选择性地从用于制备具有易位能力的蛋白脂质体的粗糙ER膜的洗涤剂提取物中去除p180。从p180耗尽提取物制备的蛋白脂质体显示减少核糖体结合水平的胰蛋白酶灭活的控制，以及在他们的能力coconferentially易位两个不同的分泌蛋白前体的损失。当纯化的p180蛋白脂质体形成之前，添加回耗尽提取物，核糖体结合和易位活性恢复。此外，单克隆抗体以及它们的Fab'片段在与完整的粗糙微粒体结合时能够抑制核糖体结合和蛋白质易位。这些数据提供了直接的证据表明，180 kD的核糖体受体是必不可少的核糖体结合和新生蛋白质的易位穿过粗糙的ER膜。
We have previously isolated a 180-kD ribosome receptor (p180) from mammalian rough ER that, when incorporated into liposomes, bound ribosomes with an affinity similar to intact membranes. To directly assess the contribution of p180 to ribosome binding as well as protein translocation, monoclonal antibodies were used to selectively deplete p180 from the detergent extracts of rough ER membranes used in the preparation of translocation-competent proteoliposomes. Proteoliposomes prepared from p180-depleted extracts showed a reduction in ribosome binding to the level of trypsin-inactivated controls as well as a loss in their ability to cotranslationally translocate two different secretory protein precursors. When purified p180 was added back to depleted extracts before proteoliposome formation, both ribosome binding and translocation activity were restored. In addition, the monoclonal antibodies, as well as their Fab' fragments, were able to inhibit ribosome binding and protein translocation when bound to intact rough microsomes. These data provide direct evidence that the 180-kD ribosome receptor is essential for ribosome binding and for the translocation of nascent proteins across the membrane of the rough ER.