The Role of Phosphatidylcholine Biosynthesis in the Regulation of the INO1 Gene of Yeast

Peter Griac,Marci J Swede,Susan A Henry

doi:10.1074/jbc.271.41.25692

Peter Griac, Marci J Swede + Show 1 more

Open Access

https://doi.org/10.1074/jbc.271.41.25692

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Abstract

In yeast, as in other eukaryotes, phosphatidylcholine (PC) can be synthesized via methylation of phosphatidylethanolamine or from free choline via the CDP-choline pathway. In yeast, PC biosynthesis is required for the repression of the phospholipid biosynthetic genes, including the INO1 gene, in response to inositol. In this study, we analyzed the effect of mutations in genes encoding enzymes involved in PC biosynthesis on the transcriptional regulation of phospholipid biosynthetic genes. We report that repression of INO1 transcription in response to inositol is clearly dependent on ongoing PC biosynthesis, but it is independent of the route of synthesis. Our results also suggest that intermediates in the phosphatidylethanolamine methylation and CDP-choline pathways are not responsible for generating the regulatory signal that results in repression of INO1 and other coregulated genes of phospholipid biosynthesis. Furthermore, repression of INO1 is not tightly correlated to the proportion of PC in the total cellular phospholipids. Rather, we report that when the rate of synthesis of PC becomes growth limiting, the addition of inositol fails to repress the phospholipid biosynthetic genes, but when the rate of PC synthesis is sufficient to sustain normal growth, the addition of inositol to the growth medium has the effect of repressing INO1 and other phospholipid biosynthetic genes.

Highlights

Phosphatidylcholine is synthesized in eukaryotic cells via two distinct pathways
Genes that have been shown to exhibit this pattern of transcriptional regulation include INO1, CKI1, CPT1, CHO1, CHO2/PEM1, and OPI3/PEM2 (Fig. 1)
INO1 regulation is restored in cho1 mutants if ethanolamine, monomethylethanolamine, dimethylethanolamine, or choline is supplied [13]

Summary

EXPERIMENTAL PROCEDURES

Materials—Sources of materials were: [32P]orthophosphate (carrier free), [␣-32P]cytidine 5Ј-triphosphate (specific activity, 800 Ci/mmol), [methyl-14C]choline chloride (specific activity, 40 – 60 mCi/mmol), DuPont NEN; SG81 paper, GF/A glass fiber filters, Whatman; nitrocellulose, Schleicher & Schull; SP6/T7 Transcription Kit, Boehringer Mannheim. To block the CDP-choline pathway, we used a strain in which the CKI1 gene was disrupted, as well as a double mutant strain carrying CPT1 and EPT1 gene disruptions simultaneously. To construct a CHO2 disruption in the genetic background of cpt, ept, the SalI-BglII internal fragment of the CHO2 gene [11, 15], carried on plasmid pSPT18 (Boehringer Mannheim), was replaced with the TRP1 gene. Stable TRPϩ transformants were tested for the characteristic cho mutant inositol excretion (OpiϪ) phenotype [11, 12], and the disruption of a CHO2 gene was confirmed by the absence of CHO2 mRNA on Northern blots In this way, we obtained a set of isogenic strains with blocks in the methylation pathway, the CDP-choline pathway, or simultaneously in both pathways. DBY746 DBY746 cho HJ000 HJ000 cho CTY393 w303–1B DC5 SH335 SH336 (diploid) 1–9A 1–9B 1–9C 1–9D

TABLE I Yeast strains

RESULTS

IϩCϩ IϩCϪ IϩCϩ IϩCϪ

DISCUSSION