The AAPT1 gene of soybean complements a cholinephosphotransferase-deficient mutant of yeast.
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 14C-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.
Dewey
R E
RE
Department of Crop Science, North Carolina State University, Raleigh 27695-7620.
Wilson
R F
RF
Novitzky
W P
WP
Goode
J H
JH
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003565:Cytidine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D018076:DNA, Complementary / Q000235:genetics; D002798:Diacylglycerol Cholinephosphotransferase / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D004982:Ethanolaminephosphotransferase / Q000172:deficiency / Q000235:genetics; D004983:Ethanolamines / Q000494:pharmacology; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant / Q000235:genetics*; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D012639:Seeds / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D017386:Sequence Homology, Amino Acid; D030262:Soybean Proteins; D013025:Glycine max / Q000201:enzymology / Q000235:genetics*; D014158:Transcription, Genetic
D018076:DNA, Complementary; D004983:Ethanolamines; D010940:Plant Proteins; D030262:Soybean Proteins; C006933:CDP ethanolamine; D003565:Cytidine Diphosphate; D004982:Ethanolaminephosphotransferase; D002798:Diacylglycerol Cholinephosphotransferase