Characterization of un-17, chol-3 and chol-4, three phospholipid biosynthetic mutants of Neurospora crassa
FGN 47:96-98

Marta Goodrich-Tanrikulu,* 1922 Rio Grande Street, Davis, CA 95616 USA

*current address: Bio-Rad Laboratories, 2000 Alfred Nobel Drive, Hercules, CA 94547 USA

Four choline-requiring mutants of Neurospora crassa have been identified genetically. The chol-1 and chol-2 mutants are impaired in phosphatidylcholine biosynthesis. Phospholipid synthesis is also impaired in the inl and un-17 mutants; the inl mutant cannot synthesize inositol phospholipids. Defects in phospholipid biosynthesis are reported here for three previously uncharacterized mutants, un-17, chol-3 and chol-4.

Phospholipid biosynthesis in fungi is best characterized in Saccharomyces cerevisiae and Neurospora crassa. Much of the early work on phospholipid synthesis relied on the biochemical and genetic characterization of the choline-requiring mutants chol-1 and chol-2, and the inositol-requiring mutants at the inl (inos) locus of Neurospora. These mutants have defects at the following points in the biosynthetic pathway (Figure 1): chol-1, the enzyme that carries out the first of the three methylation steps converting the phospholipid phosphatidylethanolamine to phosphatidylcholine; chol-2, the bifunctional enzyme that carries out the second and third methylation steps of phosphatidylethanolamine to phosphatidylcholine (Crocken and Nyc, J. Biol. Chem. 239: 1727-1730, 1964); and inl, the enzyme that converts glucose-6-phosphate to inositol-1-phosphate (Zsindely et al., Acta Biol. Acad. Sci. Hung. 28: 381-290, 1977).** The inl defect results in the inability to synthesize inositol phospholipids, including phosphatidylinositol (Shatkin and Tatum, Am. J. Bot. 48: 760-771, 1961). More recently, several laboratories have advanced the biochemical, genetic and molecular understanding of phospholipid biosynthesis in Saccharomyces, for which a large number of mutants have now been characterized. Among the genes identified are the apparent homologs of the Neurospora chol-1, chol-2, and inl genes, several genes affecting their regulation, and genes affecting earlier steps of the phosphatidylcholine biosynthetic pathway (see Carman and Zeimetz, J. Biol. Chem. 271: 13293-13296, 1996, for a brief review).

In Neurospora, one potential regulatory mutant has been described (a suppressor of inl; Giles and Partridge, Proc. Natl. Acad. Sci. USA 39: 479-488, 1953) and three additional mutants that may affect the phospholipid biosynthetic pathway are available, but have not been characterized. One of these is the temperature-sensitive un-17 mutant. This mutant is a potential mutant in phospholipid biosynthesis because its growth cessation at the restrictive temperature (34C) correlates with a dramatic reduction in phosphorus incorporation into phospholipids (Inoue and Ishikawa, Arch. Microbiol. 104: 1-6, 1975). The choline-requiring mutants chol-3 and chol-4 are also potential mutants in phospholipid biosynthesis; supplementation with choline allows biosynthesis of phosphatidylcholine by a minor pathway that utilizes CDP-choline. In Saccharomyces, mutations earlier in the phosphatidylcholine biosynthetic pathway can also lead to a choline requirement. Aside from phospholipid biosynthetic or regulatory mutants, no other choline-requiring mutants are known. These two Neurospora mutants originated from the collection of Tatum, who identified them as choline requiring (Perkins and Pollard, Fungal Genet. Newsl. 34: 52-53, 1986). The chol-4 mutant, because of its choline requirement and close linkage to chol-1, initially was suspected to be an allele of chol-1; however, chol-1 and chol-4 have recently been shown to be nonallelic (D. Perkins, pers. comm.). Phospholipid biosynthesis in un-17 and in the chol-3 and chol-4 mutants was therefore compared to wild type and to the previously characterized Neurospora phospholipid biosynthetic mutants.

Wild-type Neurospora crassa strain 74-OR23-IA (FGSC #987), the chol-1 (FGSC #2982), chol-2 (FGSC #4095), inl (FGSC #497) and un-17 (FGSC #2356) mutants were obtained from the Fungal Genetics Stock Center. The chol-3 (FGSC #4646) and chol-4 (FGSC #4648) mutants were obtained from David Perkins, as was a temperature-sensitive inl allele(FGSC #2258).

The chol-3 and chol-4 mutants grow much more slowly than wild type, chol-1, or chol-2, even in the presence of choline. Only trace amounts of phospholipids and other lipids are produced in chol-3 and chol-4 cultures (similar to unsupplemented inl or chol-1 or chol-2). Other water-soluble supplements (for example inositol, ethanolamine, monomethylethanolamine, dimethylethanolamine, and fatty acids in the form of Tween detergents) were tested for ability to better support growth of chol-3 and chol-4, and of un-17 above its restrictive temperature, without success. The chol-3 and chol-4 mutants are leaky, and chol-4 showed a tendency toward reversion to a wild-type phenotype.

To determine profiles of phospholipid synthesis relative to wild type, cultures were radiolabelled for 1 hr with 0.5 Ci of the phospholipid precursor 14C-glycerol (Moravek) in the presence of suboptimal (20 M) or no supplement at 35C. This temperature is a restrictive temperature for chol-2, for un-17, and for the temperature-sensitive inl mutant. To increase mass, cultures were grown for 1 day prior to labelling (30C), then shifted to 35C, with label added 30 min after the temperature shift. For some experiments, 14C-acetate (Amersham) was instead used as a precursor.

After radiolabelling, lipids were extracted and phospholipids separated by TLC in the solvent system 1-propanol/chloroform/propionic acid/0.1% KCl, 3/2/2/1. Lipids were stained with iodine, and label incorporation was determined by radiodensitometry. For all of the mutants, 14C-glycerol incorporation into phospholipids differed more from the wild-type profile than did 14C-acetate incorporation, most likely because fatty acid groups (derived from acetate) are exchanged relatively rapidly among different lipids, which obscures effects of a mutation at a specific point in the pathway.

The profiles of labelled precursor incorporation into phospholipids of the chol-1, the chol-2, and the inl mutant showed the expected differences relative to wild type (Figure 2). For example, the chol-2 mutant accumulated the intermediate phosphatidylmonomethylethanolamine. (The inhibition of phosphatidylinositol accumulation was greater for the temperature-sensitive inl allele than for the other inl strain grown with suboptimal inositol.) Such clear-cut results supported the use of a similar radiotracer approach to identify any biochemical defects in phospholipid synthesis of the uncharacterized mutants.

Radiotracer results with the three mutants chol-3, chol-4 and un-17 are consistent with phospholipid biosynthetic defects. Consistent with their slow growth, incorporation of labelled precursors into phospholipids of both chol-3 and chol-4 was reduced (over 1 hr, about 10% that of wild type for 14C-acetate, and 30% for 14C-glycerol). A similar overall reduction in incorporation was observed in the absence of supplementation for chol-1 and chol-2 mutants and for inl mutants, consistent with earlier reports. Slow growth may be an indirect effect of the mutations, due to grossly altered membrane phospholipid composition (Hubbard and Brody, J. Biol. Chem. 250: 7173-7181, 1975), although in Saccharomyces, inhibition of phosphatidylinositol synthesis results in negative regulation of phosphatidylcholine synthesis, and vice versa.

Based on 14C-glycerol incorporation results, un-17 appears to be defective in the phosphatidylethanolamine methylation pathway. Intensely staining bands corresponding to the methylation intermediates are visible following TLC separation of lipids from cultures shifted to the restrictive temperature. The Neurospora chol-1 and chol-2 mutants have been well characterized as having defects in the required methyltransferases. Despite extensive genetic and biochemical studies in Saccharomyces, a second subunit of the phosphatidylmonomethylethanolamine methyltransferase has not been postulated. Also, in contrast to chol-2, supplementation of un-17 with dimethylethanolamine does not restore growth. Therefore, un-17 is unlikely to be a methyltransferase mutant, unless growth at high (>34C) temperatures requires a distinct methyltransferase from that encoded by the chol-2 gene. Furthermore, to explain the inability of either dimethylethanolamine or choline to restore wild-type growth, the alternative CDP-choline biosynthetic pathway for phosphatidylcholine would also have to be impaired at high temperatures. The mutation may alternatively impair all three of the methylation steps, each of which requires S-adenosylmethionine. However, the mutant maps to a distinct locus from eth-1, the gene for S-adenosylmethionine synthase. In Saccharomyces, phospholipid biosynthetic genes are coordinately regulated, so a regulatory mutation is also a possibility, provided it has an especially pronounced effect on synthesis of phosphatidylcholine.

14C-glycerol incorporation also shows a distinct labelling pattern for chol-3, indicating that the mutant is blocked in the formation of phosphatidylinositol. In heterokaryons, chol-3 complemented inl mutants, so chol-3 is not allelic to inl. In addition, inositol supplementation did not restore wild-type growth of chol-3. These results suggest that chol-3 affects the formation of phosphatidylinositol from CDP-diacylglycerol. An alternative possibility, that a higher activity of phosphatidylinositol lipase in chol-3 accounts for the results, is unlikely given that both chol-3 and inl incorporated significant label from 14C-acetate into phosphatidylinositol, presumably by exchange of fatty acid groups from other phospholipids.

The chol-4 mutant appears to have either a partial block in the synthesis of phosphatidylserine, or a block earlier in the pathway. A regulatory mutation cannot be ruled out. Further work is thus needed to identify the nature of the chol-4 defect.

**Some biochemical evidence conflicts, suggesting an effect on the following biochemical step. However, a partial sequence of the cloned inl gene is homologous to the S. cerevisiae and other cloned myo-inositol-1-synthases, which catalyze this step (M. G.-T., unpublished data).

Special thanks to David Perkins, Hirokazu Inoue, Kevin McCluskey and Alan Radford for their interest in this investigation, and to Robert Metzenberg and an anonymous reviewer for sharing insightful comments. The author also thanks the Perkins and Yanofsky laboratories, and Calgene LLC, for use of their facilities during this study.

Figure 1. Outline of phospholipid biosynthesis in Neurospora. The major phospholipids (PI, phosphatidylinositol; PS, phosphatidylserine; PE, phosphatidylethanolamine; PC, phosphatidylcholine) are shown in bold. Also shown are the points of entry into the pathway of the soluble phospholipid precursors inositol, choline, glycerol and acetate. PMME (phosphatidylmonomethylethanolamine) and PMDE (phosphatidyl-dimethylethanolamine) are minor phospholipids that accumulate in the chol-2 mutant. Choline-supplemented chol-1 and chol-2 mutants are able to synthesize PC by the minor CDP-choline pathway, indicated by dotted arrows.

Figure 2. Incorporation of 14C-glycerol into phospholipids. Cultures were labeled for 1 hr, beginning 30 min after transfer of cultures to 35C. Data are averages s.d. for 3 or 4 cultures (1 each for chol-1 and chol-2 under these labeling conditions). In this experiment, the temperature-sensitive inl strain was used.

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