Savitz A J, Meyer D I
Department of Biological Chemistry, University of California, Los Angeles School of Medicine.
J Cell Biol. 1993 Feb;120(4):853-63. doi: 10.1083/jcb.120.4.853.
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.
我们之前从哺乳动物粗面内质网中分离出一种180-kD核糖体受体（p180），将其整合到脂质体中时，它结合核糖体的亲和力与完整膜相似。为了直接评估p180对核糖体结合以及蛋白质转运的作用，我们使用单克隆抗体从用于制备具有转运能力的蛋白脂质体的粗面内质网膜去污剂提取物中选择性去除p180。从去除p180的提取物制备的蛋白脂质体显示核糖体结合减少到胰蛋白酶灭活对照的水平，并且其共翻译转运两种不同分泌蛋白前体的能力丧失。当在形成蛋白脂质体之前将纯化的p180添加回耗尽的提取物中时，核糖体结合和转运活性均得以恢复。此外，单克隆抗体及其Fab'片段在与完整的粗面微粒体结合时能够抑制核糖体结合和蛋白质转运。这些数据提供了直接证据，表明180-kD核糖体受体对于核糖体结合以及新生蛋白质跨粗面内质网膜的转运至关重要。
