Isolation and characterization of a yeast mutant defective in cholinephosphotransferase

Abstract

A yeast mutant defective in cholinephosphotransferase (cpt) was isolated as a revertant from a choline-sensitive mutant, which exhibited lowered phosphatidylinositol synthesis. A block at the cholinephosphotransferase step in the mutant was indicated by the enzyme defect and the accumulation of CDP-choline in the cells with a decrease in phosphatidylcholine synthesis. The defect was due to a single recessive mutation in a nuclear gene. The residual activity in the mutant showed an increased apparent K, for CDP-choline and an altered sensitivity to Tween 20. Thus the structural gene may be affected in the mutant. The occurrence of an intact ethanolaminephosphotransferase in the mutant indicates the distinctness of the genes encoding cholinephosphotransferase and ethanolaminephosphotransferase in yeast. The present selection method was also effective for isolating mutants defective in the other steps of the CDP-choline pathway and choline transport.For a better understanding of cellular growth and cellcycle control, knowledge of the genetic control of phospholipid metabolism is essential. The yeast Saccharomyes cerevisiue possesses a phospholipid-synthetic pathway similar to those found in other eucaryotic cells [l]. Because of the feasibility of the genetic and moleclar-biological approaches, the yeast system seems to be particularly useful for studying the genetic control of eucaryotic phospholipid metabolism.The CDP-choline pathway [2 -41 is widely distributed in a variety of eucaryotic cells including yeast [5, 61, and forms phosphatidylcholine from choline with CDP-choline as an intermediate. The pathway involves choline kinase, cholinephosphate cytidylyltransferase and cholinephosphotransferase. Cholinephosphotransferase, which catalyzes the last step of the pathway, is of special interest, since this enzyme is involved in the branching point for the synthesis of phosphatidylcholine and triacylglycerol [4, 71. In previous studies [8, 91 yeast mutants, defective in the CDP-choline

CDPcholine:1,2‐diacylglycerol cholinephosphotransferase from rat liver microsomes. I. Solubilization and characterization of the partially purified enzyme and the possible existence of an endogenous inhibitor

“…EPT activity was purified 6-fold with 60% recovery, indicating that the latter activity remained relatively stable in the course of the solubilization-purification process. A number of reports had previously indicated that EPT, which is an enzyme distinct from CPT (25)(26)(27)(28), is much more stable than CPT in the presence of various detergents, including DOC (14,(29)(30)(31).…”

Cloning of a human choline kinase cDNA by complementation of the yeast cki mutation

“…Mutants defective only in the choline kinase gene (CKI) do not show any apparent growth phenotype [6], and are thus not suitable for ¢omplementation cloning. We introduced cki into the pss background to construct a cki pss double mutant.…”

“…DNA encoding choline kinase has been isolated from two sources, yeast and rat liver, in our laboratory [3,4]. The yeast gene was obtained by complementation of the choline kinase mutation, cki [5,6], and rat liver choline kinase eDNA was recently cloned, using specific antibodies, from a 2gtl I rat liver eDNA library. The open reading frames in the yeast and rat liver clones encoded 582 and 435 amino acid residues with relative molecular masses of 66,316 and 49,737, respectively.…”