Glycosylation can influence topogenesis of membrane proteins and reveals dynamic reorientation of nascent polypeptides within the translocon.
Glycosylation can influence topogenesis of membrane proteins and reveals dynamic reorientation of nascent polypeptides within the translocon.
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DOI：
10.1083/jcb.147.2.257
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发表时间：
1999-10-18
期刊：
影响因子：
--
通讯作者：
Spiess M
中科院分区：
文献类型：
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作者：
Goder V;Bieri C;Spiess M
哺乳动物内质网中的多跨膜蛋白的拓扑结构被认为主要由第一疏水序列决定。我们分析了含有两个冲突信号序列的一系列嵌合模型蛋白的体内插入，即，NH 2-末端和内部信号，它们中的每一个通常指导其COOH-末端的易位。当信号被超过60个残基分开时，观察到线性插入，其中第二个信号充当停止转移序列。随着间隔区的缩短，插入易位COOH末端的蛋白质比例增加，如第二个信号所示。通过测量NH 2-末端信号随后是不同长度的多肽的靶向效率来测试这是否由经由第二信号的膜靶向引起。结果表明，靶向主要由蛋白质中的第一个信号介导。最重要的是，我们发现间隔序列内的糖基化影响蛋白质的方向。这表明新生多肽可以在易位机制内重新定向，这是一个被糖基化阻断的过程。因此，膜蛋白的拓扑发生是一个动态的过程，其中紧密间隔的信号和跨膜序列的拓扑发生信息被整合。
The topology of multispanning membrane proteins in the mammalian endoplasmic reticulum is thought to be dictated primarily by the first hydrophobic sequence. We analyzed the in vivo insertion of a series of chimeric model proteins containing two conflicting signal sequences, i.e., an NH2-terminal and an internal signal, each of which normally directs translocation of its COOH-terminal end. When the signals were separated by more than 60 residues, linear insertion with the second signal acting as a stop-transfer sequence was observed. With shorter spacers, an increasing fraction of proteins inserted with a translocated COOH terminus as dictated by the second signal. Whether this resulted from membrane targeting via the second signal was tested by measuring the targeting efficiency of NH2-terminal signals followed by polypeptides of different lengths. The results show that targeting is mediated predominantly by the first signal in a protein. Most importantly, we discovered that glycosylation within the spacer sequence affects protein orientation. This indicates that the nascent polypeptide can reorient within the translocation machinery, a process that is blocked by glycosylation. Thus, topogenesis of membrane proteins is a dynamic process in which topogenic information of closely spaced signal and transmembrane sequences is integrated.