[3] Strategies for generating phospholipid synthesis mutants in yeast

[3]

PHOSPHOLIPID

SYNTHESIS

MUTANTS

IN YEAST

21

[3] S t r a t e g i e s for G e n e r a t i n g P h o s p h o l i p i d S y n t h e s i s M u t a n t s in Y e a s t B y MARCI J. SWEDE, KIMBERLY A. HUDAK, JOHN M. LOPES, and

SUSAN A. HENRY Introduction Many of the methods used for generating phospholipid biosynthesis mutants in yeast are based on methods that were developed for the isolation of similar mutants in E s c h e r i c h i a coli.1 For example, rapid colony autoradiographic screening techniques originally developed for bacteria have been adapted for the identification of yeast mutants. As a simple microorganism, yeast provides many of the technical advantages of bacterial systems. In addition, it provides the opportunity to explore the regulation of phospholipids that are unique to eukaryotes such as phosphatidylinositol (PI) and phosphatidylcholine (PC). Recent success in isolating yeast mutants defective in the synthesis of these lipids has led to important insights concerning the mechanisms by which the cell regulates the proportional synthesis of its major membrane phospholipids. Mutants Auxotrophic for Phospholipid Precursors Isolation of mutants auxotrophic for phospholipid prccursors, such as inositol and cholinc, has provcd to be very successful for identifying both structural and regulatory genc mutants in yeast (Table I). For example, mutations at the I N O I locus, 2 thc structural gene for inositol-l-phosphate synthase (Fig. I; IIPS), lead to inositol auxotrophy. Cells carrying an inol mutation, w h e n starved for inositol, rapidly cease synthesis of phosphatidylinositol (PI) and, shortly thereafter, stop dividing and die) ,4 "Inositolless" death has been used in a numbcr of fungal species 5-7 to devise cnrichmcnt procedures for thc isolation of mutants with defects in a variety of othcr cellular functions including macromolccular synthesis. 8 lw. Dowhan, thisvolume [2]. 2 M. R. Culbertsonand S. A. Henry, Genetics 80, 23 0975). 3 G. Becket and R. L. Lestcr,J. Biol. Chem. 252, 8684 (1977). 4 S. A. Hcnry, K. D. Atkinson, A. I. Kolat,and M. R. Culbertson,J. Baeteriol. 130, 472

(1977). 5 H. E. Lester and S. R. Gross, Science 139, 572 (1959). 6R. Holliday, Microb. Genet. Bull. 13, 28 (1962). 7 p. L. Thomas, Can. J. Genet. Cytol. 14, 785 (1972). 8 S. A. Henry, T. F. Donahue, and M. R. Culbertson, Mol. Gen. Genet. 143, 5 (1975). METHODS IN ENZYMOLOGY, VOL. 209

Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

22

GENERATING PHOSPHOLIPID SYNTHESIS MUTANTS

[3]

TABLE I YEAST PHOSPHOLIPID MUTANTS Mutant cct cdgl chol (pss, eth)

cki cpel cptl eaml and eam2 eptl inol ino2 and ino4 lcbl opil opi2 and opi4 opi5 petal (cho2) peru2 (opi3) pirnl and pim2 pis

Description

Refs.~

Defect in choline-phosphate cytidylyltransferase also isolated as suppressor of secl4 Defect in CDPdiacylglycerol synthase Mutations in structural gene for phosphatidylserine synthase (CDP-1,2-diacyl-sn-glycerol : L-serine O-phosphatidyltransferase, EC 2.7.8.8) Mutation in choline kinase also isolated as suppressor of secl4 Mutation resulting in constitutive expression of phospholipid biosynthetic genes Mutation in structural gene for sn-l,2-diacylglycerolcholinephosphotransferase Isolated as suppressors of ethanolamine auxotrophy of chol mutants Mutation in structural gene for sn- 1,2-diacylglycerol-ethanolaminephosphotransferase Mutation in structural gene for L-myo-inositol-1phosphate synthase (EC 5.5.1.4) Mutations in regulatory genes encoding transcriptional activators of phospholipid biosynthetic genes Defect in serine palmitoyltransferase Mutation in gene encoding negative regulator of phospholipid biosynthetic genes Mutations resulting in constitutive inositol-l-phosphate synthase expression Suppressor of ino2, ino4 double mutant Mutations in structural gene for phosphatidylethanolamine methyltransferase Mutations in structural gene for phospholipid methyltransferase Defective in phosphatidylinositolphosphate kinase and phosphatidylinositol kinase activities, respectively Defect in phosphatidylinositol synthase (CDPdiacylglycerol-inositol 3-phosphatidyltransferase [EC 2.7.8.11])

1 2 3 4-9

10, 11 12 13, 14 15, 16 17 18-20 18, 20-22 23 24 24 25 26, 27 28-30 31 32

Key to references: (1) J. Nikawa, K. Yonemura, and S. Yamashita, Eur. J. Biochem. 131, 223 (1983); (2) L. S. Klig, M. J. Homann, S. D. Kohlwein, M. J. Kelley, and S. A. Henry, J. Bacteriol. 170, 1878 (1988); (3) G. Lindegren, Y. L. Hwang, Y. Oshima, and C. C. Lindegren, Can. J. Genet. Cytol. 7, 491 (1965); (4) K. D. Atkinson, B. Jensen, A. I. Kolat, E. M. Storm, S. A. Henry, and S. Fogel, J. Bacteriol. 141, 558 (1980); (5) L. Kovac, I. Gbelska, V. Poliachova, J. Subik, and V. Kovacova, Eur. J. Biochem. l U , 491 (1980); (6) J.-I. Nikawa and S. Yamashita, Biochim. Biophys. Acta 665, 420 (1981); (7) V. A. Letts and I. A. Dawes, Biochem. Soc. Trans. 7, 976 (1983); (8) K. Kiyono, K. Miura, Y. Kushima, T. Hikiji, M. Fukushima, I. Shibuya, and A. Ohta,

[3]

PHOSPHOLIPID SYNTHESIS MUTANTS IN YEAST

23

Isolation of inositol auxotrophs (Ino-) has also resulted in the identification of several classes of mutants defective in regulation of phospholipid biosynthesis. In wild-type yeast cells, the INO1 gene is repressed 9 in response to the presence of the phospholipid precursors inositol and choline. A similar pattern of regulation has been observed for a number of enzymes of phospholipid biosynthesis in yeast) T M Strains harboring a mutation in the IN02 or I N 04 gene fail to derepress the INO1 gene even in the absence of inositol. Since basal (repressed) levels of expression of the INOI gene are insufficient to allow growth in the absence of inositol, ino2 and ino4 mutants are inositol auxotrophs. The ino2 and ino4 mutants were subsequently found to have pleiotropic defects in phospholipid biosynthesis including decreased synthesis of PC. ~5 The IN02 and IN04 genes are now known to encode positive regulators of INO1 and other coregulated structural genes (Refs. 16-18; Table I). 9 j. p. Hirsch and S. A. Henry, Mol. Cell. Biol. 6, 3320 (1986). M. A. Carson, M. Emala, P. Hogsten, and C. J. Waechter, J. Biol. Chem. 259, 6267 (1984). I1 M. J. Homann, S. A. Henry, and G. M. Carman, J. Bacteriol. 163, 1265 (1985). z2 L. S. Klig, M. J. Homann, G. M. Carman, and S. A. Henry, J. Bacteriol. 162, 1135 (1985). I3 A. M. Bailis, M. A. Poole, G. M. Carman, and S. A. Henry, Mol. Cell. Biol. 7, 167 (1987). 14 M. Greenberg, S. Hubbell, and C. Lam, Mol. Cell. Biol. 8, 4773 (1988). 15 B. S. Loewy and S. A. Henry, Mol. Cell. Biol. 4, 2479 (1984). 16G. M. Carman and S. A. Henry, Annu. Rev. Biochem. 58, 635 (1989). 17 D. K. Hoshizaki, J. E. Hill, and S. A. Henry, J. Biol. Chem. 265, 4736 (1990). is M. White, J. Lopes, and S. A. Henry, Adv. Microb. Physiol. 32, 1 (1991). to

J. Biochem. 102, 1089 (1987); (9) K. Hosaka and S. Yamashita, J. Bacteriol. 143, 176

(1980); (10) A. E. Cleves, T. P. McGee, K. Champion, M. Goebl, W. Dowhan, and V. A. Bankaitis, Cell (Cambridge, Mass.) 64, 789 (1991); (11) J. Lopes, unpublished data (1991); (12) R. H. Hjelmstad and R. M. Bell, J. Biol. Chem. 262, 3909 (1987); (13) K. Hosaka and S. Yamashita, Eur. J. Biochem. 162, 7 (1987); (14) V. Bankaitis, personal communication (19 ): (15) K. D. Atkinson, Genetics 108, 533 (1984); (16) K. D. Atkinson, Genetics 111, 1 (1985); (17) R. H. Hjelmstad and R. M. Bell, J. Biol. Chem. 263, 19748 (1988); (18) M. R. Culbertson and S. A. Henry, Genetics 80, 23 (1975); (19) T. S. Donahue and S. A. Henry, J. Biol. Chem. 256, 7077 (1981); (20) T. S. Donahue and S. A. Henry, Genetics 98, 491 (1981); (21) B. S. Loewy and S. A. Henry, Mol. Cell. Biol. 4, 2479 (1984); (22) J. P. Hirsch and S. A. Henry, Mol. Cell. Biol. 6, 3320 (1986); (23) G. B. Wells and R. L. Lester, J. Biol. Chem. 258, 10200 (1983); (24) M. Greenberg, B. Reiner, and S. Henry, Genetics 108, 19 (1982); (25) B. S. Loewy, Ph.D. thesis, Albert Einstein College of Medicine, Bronx, NY (1985); (26) S. Yamashita and A. Oshima, Eur. J. Biochem. 104, 611 (1980); (27) E. F. Summers, V. A. Letts, P. McGraw, and S. A. Henry, Genetics 120, 909 (1988); (28) M. L. Greenberg, L. S. Klig, V. A. Letts, B. S. Loewy, and S. A. Henry, J. Bacteriol. 153, 791 (1983); (29) S. Yamashita, A. Oshima, J.-I. Nikawa, and K. Hosaka, Eur. J. Biochem. 128, 589 (1982); (30) P. McGraw and S. A. Henry, Genetics 122, 317 (1989); (31) I. Uno, K. Fukamaki, H. Kato, T. Takenawa, and T. Ishikawa, Nature (London) 333, 188 (1988); (32) J.-I. Nikawa and S. Yamashita, Eur. J. Biochem. 125, 445 (1982).

2,*

GENERATING PHOSPHOL1PID SYNTHESIS MUTANTS E

MME

P-E

[3] DME

¢

PA

PS

CDP-DG (

~

PE

P-C

ccT

CDP-C

CDP-E

CH01 (PSS)/

C

PDME ~ PMME OPI3 CHO2

~ PC OPI3

(ERR1)

PIS ~ k Pi

(

I

I- I-P INO1 G-6-P

Phosphatidic acid PA CDP-DG CDP-diacylglycerol Phosphatidylserine Phosphatidylethanolamine PE PMNN Phosphatidylmonomethylethanolamine PDME Phosphatidyldimethylethanolamine Phosphatidylcholine Phosphatidylinositol PI Inositol 1-phosphate I-1-P Glucose 6-phosphate G-6-P Phosphocholine P-C Phosphoethanolamine P-E

Structural Genes and Products Encoded

INOI CHOI(PSS) oPI3~l~2) cm CCT CPT1 EPT1 PIS

Inositol-l-PhosphateSynthase PhosphatidylserineSynthase PhosphatidylethanolamineMethyltransferase PhospholipidMethyltransferase Choline Kinase Cholinephosphate Cytidylyltransfemse al~-1,2-Diacylglyce rol Cholinephosphotransferase ~l,2-Diacylglycerol Ethanolaminephosphotransferase Phosphatidylinositol Synthase

FIG. 1. Pathway of phospholipid biosynthesis in Saccharomyces cerevisiae. The designations for structural genes are given alongside the relevant reactions. When more than one gene designation has been used in the literature, the designation used in this chapter is given first with other designations listed in parentheses.

[3]

PHOSPHOLIPID SYNTHESIS MUTANTS IN YEAST

25

The pis mutant requires inositol at concentrations of 100 /zM or greater, ~9 whereas the inol mutants can grow in the presence of 10/zM inositol. 2° The PISI gene reportedly encodes PI synthase (Fig. 1), and the requirement of the pis mutant for high levels of inositol is believed to be due to an alteration in the apparent Km of PI synthase for inositol. J9 In addition, Uno et al. 21 isolated mutants (piml and pim2; Table I) that are supersensitive to PI 1-phosphate antibody introduced into the cell by electroporation. The mutants appear to be defective in PI and PI-phosphate kinase activity. Inositol auxotrophy is also a common phenotype of mutants with global defects in transcription. For example, mutants with defects in the large subunit of RNA polymerase II are inositol auxotrophs owing to a failure to derepress the INO1 gene.22 Similar Ino- phenotypes have been reported for mutants with defects in a variety of other genes involved in RNA transcription. 23 However, in each of these cases, the defects in transcription involve many functions in addition to the expression oflNO1 that are unrelated to phospholipid biosynthesis. In contrast, the defects in the in02 and ino4 mutants appear to be confined to phospholipid biosynthesis 17(M. Nikoloff, personal communication, 1991). Cells unable to grow in the absence of choline and/or ethanolamine define a single genetic locus designated CH01 (PSS) (Table 1). 24-29 Letts and Dawes used a density centrifugation procedure as an enrichment for mutants defective in membrane biogenesis in the isolation of the chol mutants. 28 The CH01 gene encodes phosphatidylserine (PS) synthase (CDPdiacylglycerol-serine O-phosphatidyltransferase), 3° the enzyme that catalyzes the synthesis of PS (Fig. 1). The chol mutant strains grow when supplemented with ethanolamine or choline, synthesizing PE (or PC) from 19J.-I. Nikawa and S. Yamashita, Eur. J. Biochem. 125, 445 (1982). 20 M. R. Cuibertson, T. F. Donahue, and S. A. Henry, J. Bacteriol. 126, 243 (1976). 21 I. Uno, K. Fukamaki, H. Kato, T. Takenawa, and T. Ishikawa, Nature (London) 333, 188 (1988). 22 M. L. Nonet and R. A. Young, Genetics 123, 715 (1989). 23 K. T. Arndt, C. A. Styles, and G. R. Fink, Cell (Cambridge, Mass.) 56, 527 (1989). 24 G. Lindegren, Y. L. Hwang, Y. Oshima, and C. C. Lindegren, Can. J. Genet. Cytol. 7, 491 (1965). 25 K. D. Atkinson, B. Jensen, A. I. Kolat, E. M. Storm, S. A. Henry, and S. Fogel, J. Bacteriol. 141, 558 (1980). 26 L. Kovac, I. Gbelska, V. Poliachova, J. Subik, and V. Kovacova, Eur. J. Biochem. 111, 491 (1980). 27 J.-I. Nikawa and S. Yamashita, Biochim. Biophys. Acta 665, 420 (1981). 28 V. A. Letts and I. A. Dawes, Biochem. Soc. Trans. 7, 976 (1983). 29 V. A. Letts and S. A. Henry, J. Bacteriol. 163, 560 (1985). 30 K. Kiyono, K. Miura, Y. Kushima, T. Hikiji, M. Fukushima, I. Shibuya, and A. Ohta, J. Biochem. (Tokyo) 102, 1089 (1987).

26

GENERATING PHOSPHOLIPID SYNTHESIS MUTANTS

[3]

free ethanolamine (or choline3J), thus bypassing PS as a precursor (Fig. 1). The tightest chol mutants synthesize no PS, suggesting that yeast cells can grow and function without PS as a structural component of membranes. 25 In contrast, it was necessary to isolate mutants of E. coli defective in PS synthase as conditional lethals. 32 In other fungi such as Neurospora, 33mutants defective in the phospholipid N-methyltransferases that catalyze the reaction series phosphatidylethanolamine (PE) ~ phosphatidylmonomethylethanolamine(PMME) phosphatidyldimethylethanolamine (PDME) --->phosphatidylcholine (PC) were isolated as choline auxotrophs. In addition, pem134 and pem2 35 mutants (Table I) were isolated as choline auxotrophs and found to be defective in PE methyltransferase and phospholipid (PL) methyltransferase activity, respectively (Fig. 1). However, the opi3 (Table I) mutants have biochemical defects similar to pem2 mutants but are not choline auxotrophs.36 Likewise, cho2 mutants (Table I) which have biochemical defects identical to peml mutants are not auxotrophic for choline.37 The cho2 and opi3 mutants also exhibit an inositol excretion phenotype (Opi-) which will be discussed in detail subsequently. Genes complementing the peml, peru2, 38 cho2 37, and opi3 36 m u t a n t s were isolated independently. The restriction map of CH02 is identical to that of PEMI, and that of OPI3 is identical to P E M 2 . 36"37The cho2 and opi3 gene disruption mutants are not auxotrophic for choline and have phenotypes identical to the original cho2 and opi3 point mutants. 36'37 The tightest opi3 mutants including the gene disruptant make virtually no PC but completely substitute PMME and some PDME for PC in their membranes. 36The cho2 mutants including the gene disruptants synthesize reduced but detectable PC. 37 It is believed that the residual PL methyltransferase activity in cho2 mutants (corresponding to the gene product of the OPI3/PEM2 gene) is capable of catalyzing the conversion of PE to PMME, although at a low efficiency. The presence of PMME and PDME in opi3 strains and the remaining PC in cho2 strains is believed to account for the lack of choline auxotrophy. It has also been possible to isolate mutant strains defective in the synthesis of sphingosine-containing lipids by isolating mutants auxotrophic for sphingosine. The lcbl mutant (Table I) strains were identified on 31 E. P. Kennedy and S. B. Weiss, J. Biol. Chem. 222, 193 (1956). 32 C. R. H. Raetz, Microbiol. Reo. 42, 614 (1978). 33 G. Scarborough and J. Nyc, J. Biol. Chem. 242, 238 (1967). 34 S. Yamashita and A. Oshima, Eur. J. Biochem. 104, 611 (1980). 35 S. Yamashita, A. Oshima, J.-I. Nikawa, and K. Hosaka, Eur. J. Biochem. 128, 589 (1982). 36 p. McGraw and S. A. Henry, Genetics 122, 317 (1989). 37 E. F. Summers, V. A. Letts, P. McGraw, and S. A. Henry, Genetics 120, 909 (1988). 38 T. Kodaki and S. Yamashita, J. Biol. Chem. 262, 15428 (1987).

[3]

PHOSPHOLIPID SYNTHESIS MUTANTS IN YEAST

27

the basis of a growth requirement for 200 mM DL-erythrodihydrosphingosine 39 and lack serine palmitoyltransferase activity. Mutants

That Overproduce

Inositol

The technique of isolating analog-resistant or analog-sensitive mutants has been used quite successfully in identifying regulatory mutants defective in various metabolic processes in yeast, including amino acid biosynthesis. 4°'41Initially, mutants constitutive for I1PS were sought by screening for mutants resistant to chemical analogs ofinositol. However, since none of the 18 inositol analogs tested had any effect on the growth of wildtype yeast strains f a another strategy had to be developed for identifying mutants constitutive for I1PS expression. Mutants that express I1PS constitutively were ultimately identified using a novel bioassay for inositol excretion. 42 The mutants possessing the inositol excretion phenotype are called Opi- (for ove~roduction of inositol). The assay involves growing inositol prototrophic strains that are to be tested on agar plates in small, equally spaced patches on inositol-deficient medium. The plates are incubated for several days (preincubation period) and are then sprayed with an even lawn of the inositol-requiring tester strain (Fig. 2). The tester is a diploid strain homozygous for inol and adel mutations and is therefore an inositol auxotroph and phenotypically red. Strains that excrete inositol are identified by spraying the plate with a light coat of an aerosol of the tester strain. The plate is allowed to dry for 5 to 10 sec. The spraying is repeated 2 or 3 times. An even coat of the tester strain is essential as the colonies may run if the plates are oversprayed. A red halo, indicating growth of the tester strain, surrounds putative Opicolonies and indicates an inositol excretion phenotype. Generally, the radius of the halo is 1 mm to I cm. However, the size of the halo is variable from trial to trial even for the same strain and can be affected by factors such as the length of preincubation, the temperature at which the plates are incubated, and the condition (age, dryness, etc.) of the plates. The original mutagenesis 42 resulted in the isolation of five inositoloverproducing mutants representing four unlinked loci: opil-opi4 (Table I). Further characterization of strains carrying opil, opi2, and opi4 mutations revealed the constitutive expression of I1PS. 43 The opi2 and opi4 39 G. B. Wells and R. L. Lester, J. Biol. Chem. 258, 10200 (1983). 4o A. Schurch, J. Miozzari, and R. Vutter, J. Bacteriol. 117, 1131 (1974). 41 A. Wolfner, D. Yep, F. Mezzenguy, and G. R. Fink, J. Mol. Biol. 96, 273 (1975). 41a M, Greenberg, Ph.D. Thesis, Albert Einstein College of Medicine, Bronx, NY (1980). 42 M. Greenberg, B. Reiner, and S. Henry, Genetics 100, 19 (1982). 43 M. Greenberg, P. Goldwasser, and S. Henry, Mol. Gen. Genet. 186, 157 (1982).

28

GENERATING PHOSPHOLIPID SYNTHESIS MUTANTS

[3l

Attach to air hose

OQ

OO

Tester strain suspended in distilled water

After 2.3 days

further i n c u b a t i o n : ~

Colonies preincubated2-3 days on medium lacking inositol are sprayedwith a lawn of the inositol-requidngtester strain

The presence of a halo indicatesa

colony excretinginositol, Opi"

FIG. 2. Diagram of the Opi- test.

mutants have weak inositol excretion phenotypes, and since only one allele of each locus has been isolated, these mutants have not been analyzed in detail. A mutant isolated in a subsequent screening for Opi- mutants 44was found to be pleiotropic and to have reduced activity of CDPdiacylglycerol synthase and was consequently named cdgl (Table I). The opil mutants express lIPS, PS synthase, and the phospholipid N-methyltransferases constitutively.12 The OPI1 gene has been isolated, sequenced, and shown to encode a specific negative regulator of the IN01 gene and other structural genes encoding enzymes of phospholipid biosynthesis 45 (Table I). The opi3 mutants 42 were later found to be defective in the final two methylations in the synthesis of P C . 46 Likewise, cho2 mutants which are L. S. Klig, M. J. Homann, S. D. Kohlwein, M. J. Kelley, and S. A. Henry, J. Bacteriol. 170, 1878 (1988). 45 M. J. White, J. P. Hirsch, and S. A. Henry, J. Biol. Chem. 266, 863 (1991). M. L. Greenberg, L. S. Klig, V. A. Letts, B. S. Loewy, and S. A. Henry, J. Bacteriol. 153, 791 (1983).

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PHOSPHOLIPID SYNTHESIS MUTANTS IN YEAST

29

defective in PE methylation37 and chol mutants which have lesions in PS synthase 29 also possess an Opi- phenotype. The inositol excretion phenotype of opi3, ch02, and chol strains is conditional; when opi3 mutants are grown in medium containing dimethylethanolamine (DME) or choline, the Opi- phenotype is eliminated. 36 Likewise, in ch02 mutants the Opi- phenotype is eliminated and PC biosynthesis is restored when monomethylethanolamine (MME), DME, or choline is added to the growth medium. 37The chol mutants exhibit an Opi- phenotype that is eliminated when ethanolamine, MME, DME, or choline is added to the growth medium. 29In each case, only those precursors that enter the PC biosynthetic pathway downstream of the metabolic lesion in the particular mutant result in elimination of the Opi- phenotype (Fig. 1). The elimination of the Opiphenotype correlates in each case with the restoration of I N 0 1 regulation in response to inositol.9,29'36'37 Thus, the I N 0 1 gene is regulated in response to inositol only when PC or PDME biosynthesis is ongoing. The nature of the cellular signal coordinating ongoing PC (or PDME) biosynthesis with regulation in response to inositol is not yet unders t o o d . 16A8 However, it is clear that expression of the INO1 gene is a sensitive indicator of many perturbations in phospholipid metabolism in yeast. Many new Opi- mutants with a variety of conditional Opi- phenotypes have been isolated (P. McGraw, unpublished data, 1991). These include mutants that have an Opi- phenotype unless supplemented with MME or choline, but not DME (as well as mutants that lose the Opiphenotype when supplemented with serine). In addition, mutants with a temperature-sensitive Opi- phenotype have been isolated. These mutants presumably represent additional genes involved in phospholipid biosynthesis and/or its regulation, and they are currently undergoing further genetic and biochemical analysis (M. Swede, unpublished data, 1991). Colony Autoradiography The colony autoradiography technique has been successfully used in isolating mutants of phospholipid biosynthesis in E. coli, 47 and it has been adapted for use in yeast. 4s This technique provides a direct means of screening for mutants with specific biochemical defects without any prior knowledge about potential growth phenotypes. The technique involves recreating an enzymatic activity in situ in permeabilized colonies immobilized on a filter paper. The assay conditions will, of course, depend on the enzyme to be tested. Colonies are replica plated onto Whatman (Clifton, 4~ C. R. H. Raetz, Proc. Natl. Acad. Sci. U.S.A. 72, 2274 (1975). M. J. Homann and G. M. Carman, Anal. Biochem. 135, 447 (1983).

30

GENERATING PHOSPHOLIPID SYNTHESIS MUTANTS

[3]

N J) No. 42 paper and frozen at - 70° for 1 hr. They are then air dried to promote permeabilization. Filters are incubated in petri dishes containing the radiolabeled substrate. The reaction is quenched and the products precipitated by transferring the filters to a solution containing trichloroacetic acid (TCA). The filters are washed with cold TCA and air dried. The presence of the radiolabeled product is visualized by autoradiography. The filters are then stained with Coomassie blue for comparison with the autoradiograms. Hjelmstad and Bell49'5° used a modification of the colony autoradiograph technique to isolate yeast mutants defective in sn-l-diacylglycerol-cholinephosphotransferase (CPT) activity and sn-l-diacylglycerol-ethanolaminephosphotransferase activity (EPT). The mutants defective in CPT activity fell into three complementation groups. The CPT1 locus is believed to be the structural gene for sn-l-diacylglycerol-cholinephosphotransferase. 9 Mutants defective in EPT activity fell into five complementation groups. The EPT1 locus is believed to be the structural gene for sn-l-diacylglycerol-ethanolaminetransferase. 5° The cptl and eptl mutants have no detectable auxotrophy or other growth phenotype, indicating the value of using colony autoradiography when screening for mutants defective in specific biochemical reactions.

Suppressors

Suppressors of a mutant phenotype mapping to a second locus may represent several types of mutational events. Some of the most interesting suppressors result from mutations in proteins that interact with the protein encoded by the locus represented by the original mutation. It is presumed that suppression by this mechanism involves interaction of the two mutant proteins in such a fashion that the original defect is compensated by mutation in the second protein. Another potential suppression mechanism involves creation of a biochemical or physiological bypass of the mutated function. A suppressor analysis may provide unexpected opportunities to define relationships among cellular processes. For example, the yeast SEC14 gene was recently found to encode the PI transfer protein. 5z Temperaturesensitive secl4 mutants are blocked in transport through the Golgi complex. Thus, phospholipid transfer appears necessary for secretion in yeast. Furthermore, the secl4 null mutants are inviable, suggesting that the 49 R. H. Hjelmstad and R. M. Bell, J. Biol. Chem. 262, 3909 (1987). R. H. Hjelmstad and R. M. Bell, J. Biol. Chem. 263, 19748 (1988). 51 V. A. Bankaitis, J. R. Aitken, A. E. Cleves, and W. Dowhan, Nature (London) 34/, 561 (1990).

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PHOSPHOLIPID SYNTHESIS MUTANTS IN YEAST

31

function of the phospholipid carrier protein is indispensable to the yeast cell: 2 There is evidence that certain mutants that suppress the temperature-sensitive phenotype and the secretory defect of the secl4 mutant have defects that lie within genes defining steps in the CDP-choline pathway for PC biosynthesis. 53 These suppressor strains have defects in the incorporation of 14C-choline into PC via this pathway: 3 It has been determined that in one such suppressor mutant, the mutation lies within the CKI locus that encodes choline kinase that catalyzes the initial step of the CDPcholine pathway (Fig. 1). 53 Furthermore, a second suppressor mutation appears to be linked to the CCT locus that encodes cytidylyltransferase, the enzyme that catalyzes the second step in the CDP-choline pathway (Fig. l) (V. Bankaitis, personal communication, 1991). The secl4 null mutation is suppressed by the cptl and cki mutations, and null mutations of the cptl and cki loci suppress the secl4 gene disruption mutation, indicating that the mechanism of suppression involves bypass of secl4 function. Thus, it appears that the function of the PI transfer protein is essential only if the Kennedy pathway for PC biosynthesis is functional. However, eptl, cho2, and opi3 mutations do not suppress the secl4 phenotype. Thus, the secl4 phenotype is suppressed by some defects in PC biosynthesis, but not by others. It is thought that this phenomenon may be related to a requirement to maintain the relative concentration of certain phospholipids within localized regions of the Golgi apparatus or endoplasmic reticulum (V. Bankaitis and T. McGee, personal communication, 1991). Another example of bypass suppression in phospholipid biosynthesis involves suppression of chol ethanolamine/choline auxotrophy. Atkins o n 54,55 observed that mutations at two loci (eaml and earn2) unlinked to CHO! could alleviate the auxotrophy of chol mutants without restoring PS biosynthesis. It is believed that the eaml and earn2 mutations result in excess turnover or degradation of sphingolipids, thereby liberating sufficient ethanolamine to support growth in the absence of PS biosynthesis. Currently, an analysis of suppressors of ino2 and ino4 inositol auxotrophic phenotypes is underway. A number of second-site suppressors that alleviate the inositol auxotrophy of specific alleles of each locus have been isolated (M. Nikoloff and J. Ambroziak, personal communication, 1991). In addition, dominant mutants mapping to a single locus, OPI5, have been found to bypass the requirement for IN02 and IN04 in the derepression 52 V. A. Bankaitis, D. E. Malehorn, S. D. Emr, and R. J. Greene, J. Cell Biol. 108, 1271 (1989). $3 A. E. Cleves, T. P. McGee, K. Champion, M. Goebl, W. Dowhan, and V. A. Bankaitis, Cell (Cambridge, Mass.) 64, 789 (1991). 54 K. D. Atkinson, Genetics 108, 533 (1984). 55 K. D. Atkinson, Genetics 111, 1 (1985).

32

GENERATING PHOSPHOLIPID SYNTHESIS MUTANTS

[3]

of the INO1 structural gene.56 All of these suppressor mutants are currently undergoing further genetic and biochemical analysis. Strategies Involving Molecular Biology Gene disruptions, gene fusions, and in vitro mutagenesis have all been used to generate yeast phospholipid biosynthetic mutants. The construction of gene disruptions has been useful in the production of null mutations in cloned genes. This technique has been used to examine the phenotype of null mutations in many of the phospholipid biosynthetic genes. The onestep gene disruption method of Rothstein 57 was used to create insertion alleles of the CH01,13 CH02,37 0Pi3,36 and OPI145genes. In addition, null alleles of INO1, 58 IN04,17 IN02 (M. Nikoloff, unpublished data, 1991), and OPI145 were constructed by replacement of coding sequences with a selectable marker. 57 In each of these cases, the gene disruption or null mutants had phenotypes identical to the original point mutants. A fusion between the promoter of the INOI gene of Saccharomyces cerevisiae and the lacZ reporter gene ofE. coli has been used successfully in the identification of phospholipid biosynthetic regulatory mutants. In this system, the INO1 5' flanking region and part of its coding sequence were fused in frame to the E. coli lacZ gene. 59 This fusion was integrated in single copy at the ura3 genomic locus (Fig. 3). The INOl-lacZ fusion is regulated in response to the soluble phospholipid precursors inositol and choline in a fashion identical to the regulation of native INO1 gene. Wildtype cells containing the fusion construct are blue when grown on X-Gal medium lacking inositol and choline (derepressing condition) and white on X-Gal medium containing inositol and choline (repressing condition). Colonies that exhibit altered regulation of the INOl-lacZ fusion can be readily detected by visual screening for altered colony color on X-Gal plates. Using this strategy, mutants that are unable to repress the fusion construct have been isolated. The mutants isolated remain blue on X-Gal medium even when inositol and choline are present in the medium. The advantage of this screening procedure is that it relies on a sensitive phenotype directly related to INO1 expression. Previous screening procedures such as the search for inositol auxotrophs and Opi- mutants, required gross under- or overexpression of the INO1 gene. For example, opil mutants overexpress lIPS and the IN01 gene 2- to 3-fold. In contrast, the screening employing the gene fusion allows isolation of a class of mutants that simply fail to repress the INO1 gene but do not overexpress the 56 B. S. Loewy, Ph.D. Thesis, Albert Einstein College of Medicine, Bronx, NY (1985). 57 R. J. Rothstein, this series, Vol. 101, p. 202. 58 M. Dean-Johnson and S. A. Henry, J. Biol. Chem. 264, 1274 (1989). 59 j. M. Lopes, J. P. Hirsch, P.A. Chorgo, K. L. Schulze, and S. A. Henry, Nuc. Acid Res. 19, 1687 (1991).

[3]

33

PHOSPHOLIPID SYNTHESIS MUTANTS IN YEAST

I-C-

X.galI+C+

Blue

White

I EMS mutagenesis 30 % survival 20,000 colonies

~Chr

I

?

cpel

•

Chr X

INO1

•

I-C-

INOl-lacZ • ura3::URA3

Blue

~ChrV

X-gal I+C+ Blue

FIG. 3. Schematic representation of a mutagenic strategy used to isolate cpeI mutant strains. A strain harboring a wild-type copy of an INOl-lacZ fusion gene integrated at the ura3 locus was mutagenized with ethylmethane sulfonate (EMS) to 30% survival. The parental strain grows as a blue colony on X-Gal media (derepressed conditions; lacking inositol and choline; I - C - ) unless supplemented with 75/~M inositol and 1 mM choline (repressing conditions; I + C +), in which case it grows as white colonies. Mutants were identified that grow as blue colonies on X-Gal I + C + media and designated cpel (constitutive phospholipid expression).

gene compared to wild-type derepressed levels. Three recessive mutations (cpel for constitutive phospholipid expression) possessing this phenotype have been identified (Table I). The three mutants, belonging to a single complementation group, are currently under investigation (J. Lopes and K. Hudak, unpublished data). Media

The growth media used are as follows.

Synthetic Defined Medium 2% Glucose (w/v) 0.67% Vitamin-free yeast base (w/v) (Difco)6° Adenine I0 mg/liter, uracil 10 mg/liter 6o Difco Vitamin-free yeast base has been discontinued. All other Difco yeast base formulas contain inositol. In the future, it will be necessary to make inositol-free medium completely from scratch using the above vitamins plus ammonium sulfate, salts and trace elements following the recipe for reconstituting Difco medium as previously described?

34

GENERATING

PHOSPHOLIPID

SYNTHESIS

MUTANTS

[4]

Amino acids (lysine 20 mg/liter, arginine l0 mg/liter, leucine l0 mg/ liter, methionine l0 mg/liter) Vitamins (biotin 2/~g/liter, calcium pantothenate 400 ~g/liter, folic acid 2 /~g/liter, niacin 400 t~g/liter, p-aminobenzoic acid 200/~g/ liter, pyridoxine hydrochloride 400/~g/liter) Inositol 50-75/~M Choline 1 mM as needed YEPD Medium 1% Yeast extract (w/v) 2% Peptone (w/v) 2% Glucose (w/v) Conclusion The isolation of mutants defective in various aspects of phospholipid biosynthesis has provided some important insights into the regulation of phospholipid metabolism in yeast. In particular, the pleiotropic phenotypes of regulatory mutants have revealed that many of the enzymes of phospholipid biosynthesis are under common genetic control in S. cerevisiae. In addition, many mutants possess phenotypes such as overproduction of inositol that would never have been predicted in advance. Furthermore, it is possible to produce mutant yeast cells completely lacking major phospholipids that were assumed to be essential. These results illustrate the value of mutant analysis in the study of major metabolic pathways.

[4] S t r a t e g i e s for I s o l a t i n g S o m a t i c Cell M u t a n t s D e f e c t i v e in L i p i d B i o s y n t h e s i s By RAPHAEL A. ZOELLER and CHRISTIAN R. H. RAETZ Introduction Animal cells contain a wide variety of lipid molecular species. Little is known about the functions of specific lipids or how their levels are regulated within particular membranes. Most of the enzymes involved in phospholipid biosynthesis in animal cells have not been isolated or characterizcd, because of their relatively low abundance and membrane association (making them difficult to work with). The availability of animal cell mutants METHODS IN ENZYMOLOGY, VOL. 209

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