The transmembrane segment of the human transferrin receptor functions as a signal peptide.
The transmembrane segment of the human transferrin receptor functions as a signal peptide.
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DOI：
10.1002/j.1460-2075.1986.tb04395.x
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发表时间：
1986-07
期刊：
影响因子：
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通讯作者：
M. Zerial;P. Melançon;C. Schneider;H. Garoff
中科院分区：
文献类型：
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作者：
M. Zerial;P. Melançon;C. Schneider;H. Garoff
人转铁蛋白受体（TR）是包含760个氨基酸残基的蛋白质，其N末端朝向细胞质跨越膜一次。它是在没有可裂解的信号肽的情况下合成的。我们已经测试了负责其膜插入的信号是否存在于其跨膜肽内，使用组合的重组DNA/体外翻译方法。首先从重叠的TR cDNA克隆中重建完整的TR编码区，然后将其工程化到基于SP 6的转录载体中。在体外转录和随后的翻译在粗糙的微粒体的存在下产生TR分子，糖基化和正确插入到膜中。两种实验证明TR多肽的跨越区含有跨粗面内质网膜转运的信号。首先，我们删除了TR的跨越区，并表明这种缺失突变体不能插入。其次，我们发现，当TR跨膜片段被添加到它们的N末端时，两种细胞质蛋白（小鼠二氢叶酸还原酶和黑猩猩α珠蛋白）可以以预期的方向插入微粒体膜。因此，跨越肽被证明是必要的和足够的链易位。进一步的分析表明，易位事件依赖于信号识别颗粒。
The human transferrin receptor (TR) is a protein comprising 760 amino acid residues that spans the membrane once with its N terminus towards the cytoplasm. It is synthesized without a cleavable signal peptide. We have tested whether the signal responsible for its membrane insertion is present within its transmembrane peptide using a combined recombinant DNA/in vitro translation approach. The complete TR coding region was first reconstructed from overlapping TR cDNA clones and then engineered into an SP6‐based transcription vector. In vitro transcription and subsequent translation in the presence of rough microsomes yielded TR molecules that were glycosylated and correctly inserted into the membrane. Two kinds of experiments demonstrated that the spanning region of the TR polypeptide contained the signal for translocation across the membrane of the rough endoplasmic reticulum. First, we deleted the spanning region of TR and showed that this deletion mutant could not be inserted. Second, we showed that two cytoplasmic proteins (the mouse dihydrofolate reductase and the chimpanzee alpha‐globin) could be inserted into the microsomal membrane in the expected orientation when the TR transmembrane segment was added to their N termini. Thus, the spanning peptide was shown to be both necessary and sufficient for chain translocation. Further analyses demonstrated that the translocation event was dependent on the signal recognition particle.