The NH2 terminus of preproinsulin directs the translocation and glycosylation of a bacterial cytoplasmic protein by mammalian microsomal membranes.
The NH2 terminus of preproinsulin directs the translocation and glycosylation of a bacterial cytoplasmic protein by mammalian microsomal membranes.
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DOI：
10.1083/jcb.103.6.2263
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发表时间：
1986-12
期刊：
影响因子：
--
通讯作者：
Shields D
中科院分区：
文献类型：
--
作者：
Eskridge EM;Shields D
为了研究多肽激素前体中假定的分选结构域，我们构建了前胰岛素原（ppI）的氨基末端和细菌胞质酶氯霉素乙酰转移酶（CAT）之间的融合蛋白。我们的目的是确定序列的ppI，而不是信号肽，这是必要的介导细胞内的排序和分泌的细菌酶。在这里，我们描述了在体外翻译的mRNA编码两个嵌合分子含有71和38个残基，分别融合到完整的CAT序列的ppI NH 2末端。ppI信号肽和B链的14个残基足以指导CAT移位和分离到微粒体膜囊泡中。此外，CAT酶进行N-连接的糖基化，大概在一个单一的隐蔽位点，与在体外合成的天然糖蛋白的效率是相当的。部分氨基末端测序表明，在融合蛋白的下游序列不改变信号肽酶的特异性，因此，在完全相同的网站发生的ppI信号肽的裂解在天然前体。这与在原核系统中发现的结果相反。这些数据表明，ppI的前38个残基编码所有必需的信息结合到内质网膜，易位，和蛋白水解（信号序列）处理。
To investigate putative sorting domains in precursors to polypeptide hormones, we have constructed fusion proteins between the amino terminus of preproinsulin (ppI) and the bacterial cytoplasmic enzyme chloramphenicol acetyltransferase (CAT). Our aim is to identify sequences in ppI, other than the signal peptide, that are necessary to mediate the intracellular sorting and secretion of the bacterial enzyme. Here we describe the in vitro translation of mRNAs encoding two chimeric molecules containing 71 and 38 residues, respectively, of the ppI NH2 terminus fused to the complete CAT sequence. The ppI signal peptide and 14 residues of the B-chain were sufficient to direct the translocation and segregation of CAT into microsomal membrane vesicles. Furthermore, the CAT enzyme underwent N-linked glycosylation, presumably at a single cryptic site, with an efficiency that was comparable to that of native glycoproteins synthesized in vitro. Partial amino-terminal sequencing demonstrated that the downstream sequences in the fusion proteins did not alter the specificity of signal peptidase, hence cleavage of the ppI signal peptide occurred at precisely the same site as in the native precursor. This is in contrast to results found in prokaryotic systems. These data demonstrate that the first 38 residues of ppI encode all the information necessary for binding to the endoplasmic reticulum membrane, translocation, and proteolytic (signal sequence) processing.
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