Mutations in the Saccharomyces cerevisiae opi3 Gene: Effects on Phospholipid Methylation, Growth and Cross-Pathway Regulation of Inositol Synthesis

INVESTIGATIONS

Mutations in the Saccharomyces cerevisiae opi3 Gene: Effects on Phospholipid Methylation, Growth and Cross-Pathway Regulation of Inositol Synthesis

P. McGraw and S. A. Henry
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213

We report the isolation of two new opi3 mutants by EMS mutagenesis, and construction of an insertion allele in vitro using the cloned gene. We have demonstrated that the opi3 mutations cause a deficiency in the two terminal phospholipid N-methyltransferase (PLMT) activities required for the de novo synthesis of PC (phosphatidylcholine). The opi3 mutants, under certain growth conditions, produce membrane virtually devoid of PC although, surprisingly, none of the mutants displays a strict auxotrophic requirement for choline. Although the opi3 mutants grow without supplements, we have shown that the atypical membrane affects the ability of the mutant strains to initiate log phase growth and to sustain viability at stationary phase. The commencement of log phase growth is enhanced by addition of choline or to a lesser extent DME (dimethylethanolamine), and retarded by addition of MME (monomethylethanolamine). The mutant cells lose viability at the stationary phase of the cell cycle in the absence of DME or choline, and are also temperature sensitive for growth at 37{deg} especially in media containing MME. These growth defects have been correlated to the presence of specific phospholipids in the membrane. The opi3 growth defects are suppressed by an unusual mutation in the phospholipid methylation pathway that perturbs the N-methyltransferase (PEMT) activity immediately preceding the reactions affected by the opi3 lesion. We believe this mutation, cho2-S, alters the substrate specificity of the PEMT. A secondary effect of opi3 mutations is disruption of the cross pathway regulation of the synthesis of the PI (phosphatidylinositol) precursor inositol. Synthesis of inositol is controlled through regulation of the INO1 gene which encodes inositol-1-phosphate synthase. This highly regulated gene is expressed constitutively in opi3 mutants. We have used the opi3 strains to demonstrate that synthesis of either PC or PD (phosphatidyldimethylethanolamine) will restore normal regulation of the INO1 gene.

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				S. H. Loukin, C. Kung, and Y. Saimi Lipid perturbations sensitize osmotic down-shock activated Ca2+ influx, a yeast "deletome" analysis FASEB J, June 1, 2007; 21(8): 1813 - 1820. [Abstract] [Full Text] [PDF]

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				L. C. Hancock, R. P. Behta, and J. M. Lopes Genomic Analysis of the Opi- Phenotype Genetics, June 1, 2006; 173(2): 621 - 634. [Abstract] [Full Text] [PDF]

				H. A. Boumann, J. Gubbens, M. C. Koorengevel, C.-S. Oh, C. E. Martin, A. J.R. Heck, J. Patton-Vogt, S. A. Henry, B. de Kruijff, and A. I.P.M. de Kroon Depletion of Phosphatidylcholine in Yeast Induces Shortening and Increased Saturation of the Lipid Acyl Chains: Evidence for Regulation of Intrinsic Membrane Curvature in a Eukaryote Mol. Biol. Cell, February 1, 2006; 17(2): 1006 - 1017. [Abstract] [Full Text] [PDF]

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				A. G. Howe, V. Zaremberg, and C. R. McMaster Cessation of Growth to Prevent Cell Death Due to Inhibition of Phosphatidylcholine Synthesis Is Impaired at 37 {degrees}C in Saccharomyces cerevisiae J. Biol. Chem., November 8, 2002; 277(46): 44100 - 44107. [Abstract] [Full Text] [PDF]

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				C. P. Bolognese and P. McGraw The Isolation and Characterization in Yeast of a Gene for Arabidopsis S-Adenosylmethionine:Phospho-Ethanolamine N-Methyltransferase Plant Physiology, December 1, 2000; 124(4): 1800 - 1813. [Abstract] [Full Text]

				V. Contamine and M. Picard Maintenance and Integrity of the Mitochondrial Genome: a Plethora of Nuclear Genes in the Budding Yeast Microbiol. Mol. Biol. Rev., June 1, 2000; 64(2): 281 - 315. [Abstract] [Full Text] [PDF]

				K. Kim, K.-H. Kim, M. K. Storey, D. R. Voelker, and G. M. Carman Isolation and Characterization of the Saccharomyces cerevisiae EKI1 Gene Encoding Ethanolamine Kinase J. Biol. Chem., May 21, 1999; 274(21): 14857 - 14866. [Abstract] [Full Text] [PDF]

				M. I. Kanipes, J. E. Hill, and S. A. Henry The Schizosaccharomyces pombe cho1+ Gene Encodes a Phospholipid Methyltransferase Genetics, October 1, 1998; 150(2): 553 - 562. [Abstract] [Full Text]

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				K.-H. Kim, D. R. Voelker, M. T. Flocco, and G. M. Carman Expression, Purification, and Characterization of Choline Kinase, Product of the CKI Gene from Saccharomyces cerevisiae J. Biol. Chem., March 20, 1998; 273(12): 6844 - 6852. [Abstract] [Full Text] [PDF]

				J. L. Patton-Vogt, P. Griac, A. Sreenivas, V. Bruno, S. Dowd, M. J. Swede, and S. A. Henry Role of the Yeast Phosphatidylinositol/Phosphatidylcholine Transfer Protein (Sec14p) in Phosphatidylcholine Turnover and INO1 Regulation J. Biol. Chem., August 15, 1997; 272(33): 20873 - 20883. [Abstract] [Full Text] [PDF]

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