Hickman MJ, Petti AA, Ho-Shing O, Silverman S, McIsaac RS, Lee TA, Botstein D

A yeast strain lacking Met4p, the primary transcriptional regulator of the sulfur assimilation pathway, cannot synthesize methionine. We were surprised to find that this apparently simple auxotroph doesn't grow well in rich media containing excess methionine, forming small colonies on YPD plates. Faster-growing large colonies were abundant when overnight cultures ...

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were plated, suggesting that spontaneous suppressors of the growth defect arise with high frequency. To identify the suppressor mutations, we used genome-wide SNP and standard genetic analyses. The most common suppressors were loss-of-function mutations in OPI1, encoding a transcriptional repressor of phospholipid metabolism. Using a new system that allows rapid and specific degradation of Met4p, we could study the dynamic expression of all genes following loss of Met4p. Experiments using this system with and without Opi1p showed that Opi1p represses genes that maintain levels of S-adenosylmethionine (SAM), the substrate for most methyltransferase reactions. Cells lacking Met4p grow normally when either SAM is added to the media or one of the SAM synthetase genes is overexpressed. SAM is used as a methyl donor in three reactions to create the abundant membrane phospholipid, phosphatidylcholine. Our results show that rapidly growing cells require significant methylation, likely for the biosynthesis of phospholipids.

Date: Sep. 07, 2011

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