Bernhard W R, Thoma S, Botella J, Somerville C R
Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824.
Plant Physiol. 1991 Jan;95(1):164-70. doi: 10.1104/pp.95.1.164.
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.
通过用基于该蛋白质氨基酸序列的合成寡核苷酸探测文库，分离出了一个编码菠菜（Spinacia oleracea）非特异性脂质转运蛋白的cDNA克隆。DNA序列测定表明有一个354个核苷酸的开放阅读框，它编码一个118个氨基酸残基的多肽。开放阅读框的前26个氨基酸在成熟蛋白中不存在，它们具有通常与膜蛋白或分泌蛋白合成相关的信号序列的所有特征。cDNA的体外转录以及在犬胰腺微粒体或培养的玉米胚乳细胞微粒体存在下的翻译表明，前体蛋白加工成成熟形式与共翻译插入微粒体膜有关。由于没有已知的机制可使该多肽从微粒体膜转移到细胞质中，因此该蛋白在催化细胞内膜之间脂质转移中的假定作用受到质疑。尽管脂质转运蛋白是叶细胞中最丰富的蛋白质之一，但基因组Southern分析结果表明仅存在一个基因。通过Northern印迹分析mRNA水平表明，该转录本在叶和叶柄组织中的丰度比肌动蛋白转录本高几倍，但在根中的含量不到叶柄中水平的1%。
