Role of Amino-phospholipid Synthesis in Biogenesis of Endoplasmic Reticulum of Yeast

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I.Analysis of phosphatidylserine synthesis (PSS).Phosphatidylserine synthase resides in endoplasmic reticulum (ER). To investigate its structure that is necessary to be targeted to ER and maintained there, (1) deletion mutants of the CHO1 gene that encodes the enzyme were propagated by in vitro mutagenic methods. (2) Rabit antiserum against PSS was also prepared. (2) PSS protein was overproduced as a glutathione S-transferase (GST) - PSS fusion protein in Escherichia coli, then affinity-purified to prepare rabit antiserum. These materials are very useful to clarify the mechanism of assembly of PSS into the ER membrane.II.Analysis of CTP : phosphoethanolamine cytidylyltransferase (ECT).Mutants defective in ECT activity were isolated and DNA clones that complemented the mutation were isolated. Sequencing analysis of 3.2 kb DNA of the complementary region revealed a 972 bp open reading frame of which deduced amino acid sequence had a significant similarity to CTP : phosphocholine cytidylyltransferases of yeast and rat, which are the enzymes of similar function. The gene product which was expressed in E.coli as a fusion protein to GST exhibited ECT activity. We concluded that the cloned gene ECT1 codes for a structural subunit of the enzyme ECT.