THE AAPT1 GENE OF SOYBEAN COMPLEMENTS A CHOLINEPHOSPHOTRANSFERASE-DEFICIENT MUTANT OF YEAST
THE AAPT1 GENE OF SOYBEAN COMPLEMENTS A CHOLINEPHOSPHOTRANSFERASE-DEFICIENT MUTANT OF YEAST
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DOI：
10.1105/tpc.6.10.1495
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发表时间：
1994-10-01
期刊：
影响因子：
11.6
通讯作者：
GOODE, JH
中科院分区：
文献类型：
--
作者：
DEWEY, RE;WILSON, RF;GOODE, JH
氨基醇磷酸转移酶（AAPTases）利用二酰基甘油和胞苷二磷酸（CDP）-氨基醇作为底物合成丰富的膜脂质磷脂酰胆碱和磷脂酰乙醇胺。大豆cDNA编码的AAPTase，表现出高水平的CDP-胆碱：sn-1，2-二酰基甘油胆碱磷酸转移酶的活性，是通过互补的酵母菌株在这一功能缺陷，并命名为AAPT 1分离。大豆cDNA的推导的氨基酸序列显示几乎相等的相似性，从酵母的两个特征AAPTase序列，胆碱磷酸转移酶和乙醇胺磷酸转移酶（CDP-乙醇胺：sn-1，2-二酰基甘油乙醇胺磷酸转移酶）。此外，大豆AAPT 1编码的酵母微粒体膜中的酶活性的测定显示，加入CDP-乙醇胺的反应抑制C-14-CDP-胆碱掺入磷脂酰胆碱的方式非常相似，观察到使用未标记的CDP-胆碱。虽然DNA凝胶印迹分析表明，AAPT 1样序列在大豆中作为一个小的多基因家族，相同的AAPT 1亚型分离的幼叶cDNA文库也回收从发展中的种子cDNA文库。使用仅长度不同的大豆AAPT 1 cDNA在酵母中进行的表达测定表明，转录本5'前导序列中的序列负责该异源系统中基因活性的负调节。由位于AAPT 1阅读框上游124 bp的短开放阅读框介导的翻译抑制是为观察到的基因活性下调而提出的一种模型。
Aminoalcoholphosphotransferases (AAPTases) utilize diacylglycerols and cytidine diphosphate (CDP)-aminoalcohols as substrates in the synthesis of the abundant membrane lipids phosphatidylcholine and phosphatidylethanolamine. A soybean cDNA encoding an AAPTase that demonstrates high levels of CDP-choline:sn-1,2-diacylglycerol cholinephosphotransferase activity was isolated by complementation of a yeast strain deficient in this function and was designated AAPT1. The deduced amino acid sequence of the soybean cDNA showed nearly equal similarity to each of the two characterized AAPTase sequences from yeast, cholinephosphotransferase and ethanolaminephosphotransferase (CDP-ethanolamine:sn-1,2-diacylglycerol ethanolaminephosphotransferase). Moreover, assays of soybean AAPT1-encoded enzyme activity in yeast microsomal membranes revealed that the addition of CDP-ethanolamine to the reaction inhibited incorporation of C-14-CDP-choline into phosphatidylcholine in a manner very similar to that observed using unlabeled CDP-choline. Although DNA gel blot analysis suggested that AAPT1-like sequences are represented in soybean as a small multigene family, the same AAPT1 isoform isolated from a young leaf cDNA library was also recovered from a developing seed cDNA library. Expression assays in yeast using soybean AAPT1 cDNAs that differed only in length suggested that sequences in the 5' leader of the transcript were responsible for the negative regulation of gene activity in this heterologous system. The inhibition of translation mediated by a short open reading frame located 124 bp upstream of the AAPT1 reading frame is one model proposed for the observed down-regulation of gene activity.