Relationships between translation of pro alpha1(I) and pro alpha2(I) mRNAs during synthesis of the type I procollagen heterotrimer.
Relationships between translation of pro alpha1(I) and pro alpha2(I) mRNAs during synthesis of the type I procollagen heterotrimer.
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I 型前胶原异源三聚体合成过程中 pro alpha1(I) 和 pro alpha2(I) mRNA 翻译之间的关系。
DOI：
10.1002/jcb.240590211
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Veis,A
中科院分区：
文献类型：
--
作者：
Hu,G;Tylzanowski,P;Inoue,H;Veis,A
I型前胶原异源三聚体分子的最终组装是由已完成或接近完成的新生前α链的羧基前肽结构域之间的相互作用引发的。这些相互作用记录了三螺旋折叠的链。然而，在这些事件发生之前，必须将适当的新生链带入内质网（ER）的相同隔室中。我们假设，新生pro α1（I）和pro α2（I）链合成的共定位是由链合成期间其翻译复合物之间的相互作用引起的。通过研究在存在和不存在微粒体膜的情况下以及在具有合成pro α1同源三聚体或正常异源三聚体能力的细胞中，在体外翻译过程中pro α-链信息的多聚核糖体负载，对此进行了研究。将重组人pro α1（I）和pro α2（I）cDNA插入质粒中，体外转录。在无细胞兔网织红细胞裂解物±犬胰腺微粒体中单独和混合翻译所得RNA。加入环己酰亚胺（100 μg/ml），收集多核糖体，并在15-50%蔗糖梯度上分级。从每个级分中提取RNA，并通过RT-PCR测定每个链信息的水平。类似地分析了来自K16（异源三聚体产生）、W8（原α1（I）同源三聚体）和A2′（异源三聚体+同源三聚体）细胞的多聚体。pro α1（I）和pro α2（I）信息的翻译在无细胞、无膜系统中独立进行，但在膜存在下协调改变。无细胞+膜翻译系统模拟了可比较的细胞多核糖体mRNA加载分布的行为。这些数据都表明，在ER膜表面的前α链翻译复合物之间存在相互作用，其在时间和空间上定位新生链以进行有效的异聚体选择和折叠。© 1995 Wiley利斯公司
Final assembly of the procollagen I heterotrimeric molecule is initiated by interactions between the carboxyl propeptide domains of completed, or nearly completed nascent pro α chains. These interactions register the chains for triple helix folding. Prior to these events, however, the appropriate nascent chains must be brought within the same compartments of the endoplasmic reticulum (ER). We hypothesize that the co‐localization of the synthesis of the nascent pro α1(I) and pro α2(I) chains results from an interaction between their translational complexes during chain synthesis. This has been investigated by studying the polyribosomal loading of the pro α‐chain messages during in vitro translation in the presence and absence of microsomal membranes, and in cells which have the ability to synthesize the pro α1 homotrimer or the normal heterotrimer. Recombinant human pro α1(I) and pro α2(I)CDNAs were inserted into plasmids and then transcribed in vitro. The resulting RNAs were translated separately and in mixture in a cell‐free rabbit reticulocyte lysate ± canine pancreatic microsomes. Cycloheximide (100 μg/ml) was added and the polysomes were collected and fractionated on a 15–50% sucrose gradient. The RNA was extracted from each fraction and the level of each chain message was determined by RT‐PCR. Polysomes from K16 (heterotrimer‐producing), W8 (pro α1(I) homotrimer), and A2′ (heterotrimer + homotrimer) cells were similarly analyzed. Translations of the pro α1(I) and pro α2(I) messages proceeded independently in the cell‐free, membrane‐free systems, but were coordinately altered in the presence of membrane. The cell‐free + membrane translation systems mimicked the behavior of the comparable cell polysome mRNA loading distributions. These data all suggest that there is an interaction between the pro α chain translational complexes at the ER membrane surface which temporally and spatially localize the nascent chains for efficient heteromeric selection and folding. © 1995 Wiley‐Liss, Inc.