A new role for BiP: closing the aqueous translocon pore during protein integration into the ER membrane.
A new role for BiP: closing the aqueous translocon pore during protein integration into the ER membrane.
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DOI：
10.1083/jcb.200110074
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发表时间：
2002-01-21
影响因子：
7.8
通讯作者：
Johnson, Arthur E
中科院分区：
文献类型：
--
作者：
Haigh, Nora G;Johnson, Arthur E
在哺乳动物细胞中，大多数膜蛋白在称为translocons的位点互补插入ER膜。虽然每个易位子形成一个水孔，膜的渗透性屏障在整合过程中得到维持，即使当原本紧密的核糖体-易位子密封被打开以允许新生蛋白质的胞质结构域进入胞质溶胶时。为了鉴定膜完整性得以保存的机制，通过新生链中荧光探针的碰撞淬灭，在不同的整合阶段确定新生链暴露于膜的每一侧。比较完整的，空的，或BiP加载的微粒体制备的整合中间体显示，translocon孔的内腔端封闭的BiP在ATP依赖性的过程之前，细胞质核糖体-translocon密封在整合过程中打开。这种BiP功能不同于其先前确定的在关闭无核糖体、空的translocons中的作用，因为在translocon处存在核糖体和在整合过程中延伸穿过translocon孔并进入管腔的新生膜蛋白。因此，BiP是一种复杂机制中的关键组分，该机制选择性地关闭一些但不是全部由新生链占据的translocons的内腔末端。通过使用不同尺寸的碰撞淬灭剂，发现当需要BiP在整合期间密封孔时，核糖体结合的水性易位子孔的大内径收缩。因此，孔的关闭涉及易位子的大量构象变化，其与ER膜两侧涉及核糖体和BiP的结构重排的复杂序列偶联。
In mammalian cells, most membrane proteins are inserted cotranslationally into the ER membrane at sites termed translocons. Although each translocon forms an aqueous pore, the permeability barrier of the membrane is maintained during integration, even when the otherwise tight ribosome–translocon seal is opened to allow the cytoplasmic domain of a nascent protein to enter the cytosol. To identify the mechanism by which membrane integrity is preserved, nascent chain exposure to each side of the membrane was determined at different stages of integration by collisional quenching of a fluorescent probe in the nascent chain. Comparing integration intermediates prepared with intact, empty, or BiP-loaded microsomes revealed that the lumenal end of the translocon pore is closed by BiP in an ATP-dependent process before the opening of the cytoplasmic ribosome–translocon seal during integration. This BiP function is distinct from its previously identified role in closing ribosome-free, empty translocons because of the presence of the ribosome at the translocon and the nascent membrane protein that extends through the translocon pore and into the lumen during integration. Therefore, BiP is a key component in a sophisticated mechanism that selectively closes the lumenal end of some, but not all, translocons occupied by a nascent chain. By using collisional quenchers of different sizes, the large internal diameter of the ribosome-bound aqueous translocon pore was found to contract when BiP was required to seal the pore during integration. Therefore, closure of the pore involves substantial conformational changes in the translocon that are coupled to a complex sequence of structural rearrangements on both sides of the ER membrane involving the ribosome and BiP.