ISOLATION OF A CDNA CLONE FOR SPINACH LIPID TRANSFER PROTEIN AND EVIDENCE THAT THE PROTEIN IS SYNTHESIZED BY THE SECRETORY PATHWAY
ISOLATION OF A CDNA CLONE FOR SPINACH LIPID TRANSFER PROTEIN AND EVIDENCE THAT THE PROTEIN IS SYNTHESIZED BY THE SECRETORY PATHWAY
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DOI：
10.1104/pp.95.1.164
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发表时间：
1991-01-01
期刊：
影响因子：
7.4
通讯作者：
SOMERVILLE, CR
中科院分区：
文献类型：
--
作者：
BERNHARD, WR;THOMA, S;SOMERVILLE, CR
根据菠菜非特异性脂转移蛋白的氨基酸序列，利用人工合成的寡核苷酸构建文库，获得了编码该蛋白的cDNA克隆。DNA序列测定表明，该基因有一个354个核苷酸的开放阅读框，编码一个118个氨基酸残基的多肽。开放阅读框的前26个氨基酸不存在于成熟蛋白质中，它们具有信号序列的所有特征，通常与膜蛋白或分泌蛋白的合成有关。从培养的玉米胚乳细胞中获得的cDNA体外转录和在犬胰腺微体或微体存在下的翻译表明，前蛋白的蛋白分解过程与共翻译插入到微体体膜中有关。由于目前尚不清楚这种多肽是如何从微粒体膜转移到细胞质的，因此该蛋白在催化细胞内膜间脂质转移中的作用尚存疑问。虽然脂转移蛋白是叶细胞中含量最丰富的蛋白质之一，但基因组Southern分析的结果与只有一个基因的存在是一致的。Northern blotting分析表明，该转录本在叶片和叶柄组织中的丰度是肌动蛋白转录本的数倍，但在根中的含量不到叶柄的1%。
A cDNA clone encoding a nonspecific lipid transfer protein from spinach (Spinacia oleracea) was isolated by probing a library with synthetic oligonucleotides based on the amino acid sequence of the protein. Determination of the DNA sequence indicated a 354-nucleotide open reading frame which encodes a 118-amino acid residue polypeptide. The first 26 amino acids of the open reading frame, which are not present in the mature protein, have all the characteristics of a signal sequence which is normally associated with the synthesis of membrane proteins or secreted proteins. In vitro transcription of the cDNA and translation in the presence of canine pancreatic microsomes or microsomes from cultured maize endosperm cells indicated that proteolytic processing of the preprotein to the mature form was associated with cotranslational insertion into the microsomal membranes. Because there is no known mechanism by which the polypeptide could be transferred from the microsomal membranes to the cytoplasm, the proposed role of this protein in catalyzing lipid transfer between intracellular membranes is in doubt. Although the lipid transfer protein is one of the most abundant proteins in leaf cells, the results of genomic Southern analysis were consistent with the presence of only one gene. Analysis of the level of mRNA by Northern blotting indicated that the transcript was several-fold more abundant than an actin transcript in leaf and petiole tissue, but was present in roots at less than 1% of the level in petioles.