Isolation and Characterization of ECTl Gene Encoding CTP: Phosphoethanolamine Cytidylyltransferase of Saccharomyces cerevisiae

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Abstract
Sacchammyces cerevisiae mutants that were unable to utilize extracellular ethanolamine for phosphatidylethanolamine synthesis were isolated. Two of them carried recessive chromosomal mutations in a same gene and were defective in CTP:phosphoethanolamine cytidylyltransferase (ECT) activity in vitro (Ect−). In an Ect− mutant that also carried the chol mutation, phosphatidylethanolamine accounted for less than 2% of total phospholipids, suggesting the importance of ECT in phosphatidylethanolamine synthesis. By screening a genomic library on a low copy number vector, three complementary clones of different size were isolated. A 2.8-kb common DNA region carried an open reading frame (ORF) of 969 bp in length, of which a truncated form failed to complement the Ect− mutation. This ORF was identical to the previously isolated MUQ1 gene of unknown function. Its deduced amino acid sequence had significant similarity to CTP:phosphocholine cytidylyltransferases of yeast and rat. The entire ORF, when combined with the glutathione Stransferase gene and expressed in Escherichia coli, exhibited ECT activity. These results indicate that the cloned gene encodes a catalytic subunit of ECT of S. cereuisiae.

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