Inositol Metabolism in Yeasts

Inositol Metabolism in Yeasts MICHAEL J. WHITE, JOHN M. LOPES and SUSAN A. HENRY Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth A venue, Pittsburgh, P A 15213, USA

List of abbreviations . . . . . . . . . . . I. Introduction . . . . . . . . . . . 11. Biosynthesis of inositol . . . . . . . . . 111. Phosphatidylinositolbiosynthesis . . . . . . IV. Phosphatidylinositol kinases . . . . . . . . V. Role of phosphatidylinositol and phosphoinositides in yeast VI. Role of inositol in regulation of phospholipid biosynthesis . A. Regulation of inositol 1-phosphate biosynthesis . . B. Regulation of phosphatidic-acidphosphatase . . . C. Regulation of CDP-diacylglycerol synthase . . . . D. Regulation of phosphatidylglycerol phosphate synthase . E. Regulation of phosphatidylserine synthase . . . . F. Regulation of phosphatidylserine decarboxylase . . . G. Regulation of phospholipid . . _ methyltransferases . . . VII. Interconnection between phosphatidylcholine biosynthesis and regulation of phospholipid biosynthesis by inositol . . . . . . . . . . VIII. The regulatory cascade controlling IlPS and other coregulated enzymes of phospholipid synthesis . . . . . . . . . . . . . A. Positive regulators, I N 0 2 and IN04 . . . . . . . . B. Negative regulator, OPII . . . . . . . . . . . C. Epistaticinteraction of regulatory mutations . . . . . . D. Effects of the I N 0 2 , IN04 and OPII gene products on transcription of IN01 and other genes encodingphospholipid biosynthetic functions . E. A model for regulation of phospholipid synthesis by inositol and other phospholipid precursors . . . . . . . . . . . IX. Summary . . . . . . . . . . . . . . . . X. Acknowledgements. . . . . . . . . . . . . . References . . . . . . . . . . . . . . . .

ADVANCESINMlCROBlALPHYSIOLOGY,VOL. 32 ISBN 0-12421132-8

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Copyright0 1991, by Academic Press Limited All rights of reproduction in any formreserved

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MICHAEL J WHITE. JOHN M. LOPES AND SUSAN A . HENRY

List of Abbreviations

C CDP-C CDP-DG CDP-DGS Cer12P2M DAG DME del-P2GlcNAc E GlcNAc-1P transferase GPI G6P I I1P 13P 14P I1,3P2 I1,4P2 I3,4P2 11,3,4P3 11,4,5P3 I1,3,4,5P4 I1,3 ,4,6P4 IlPS MME PA PAP PC PDME PE PI PIK PI3P PI4P PIP2 PIPK PIS PMME PMTs PS

Choline CDP-choline CDP-diacylglycerol CDP-diacylglycerol synthase Ceramide (phosphoinositol)2mannose Diacylglycerol Dimethylethanolamine N-Acetylglucosaminylpyrophosphoryldolichol Ethanolamine N-Acetylglucosamine-1-phosphatetransferase GIyeerophosphatidylinositol Glucose 6-phosphate Inositol Inositol 1-phosphate Inositol 3-phosphate Inositol 4-phosphate Inositol 1,3-diphosphate Inositol 1,Cdiphosphate Inositol 3,4-diphosphate Inositol 1,3,Ctriphosphate Inositol 1,4,5-triphosphate Inositol 173,4,5-tetraphosphate Inositol 1,3,4,6-tetraphosphate Inositol-1-phosphate synthase Monomethylethanolamine Phosphatidic acid Phosphatidic-acid phosphatase Phosphatidylcholine Phosphatidyldimethylethanolamine Phosphatidylethanolamine Phosphatidylinositol Phosphatidylinositol kinase Phosphatidylinositol 3-phosphate Phosphatidylinositol 4-phosphate Triphosphorylated phosphatidylinositol Phosphatidylinositol-phosphatekinase Phosphatidylinositol synthase Phosphatidylmonometh ylethanolamine Phospholipid methyltransferases Phosphatid ylserine

INOSITOL METABOLISM IN YEASTS

PSD PSS SAM TAG PGPS

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Phosphatidylserine decarboxylase Phosphatidylserine synthase S- Adenosylmethionine Triacylgl ycerol Phosphatidylglycerophosphate synthase I. Introduction

In recent years, inositol-containing phospholipids and other metabolic products of inositol have been shown to play roles in an increasing array of vital functions in eukaryotic cells. For example, phosphoinositides have recently been implicated as second messengers in a variety of important signalling processes in mammalian cells (Michell, 1986; Majerus et al., 1986, 1988). Less information, however, is available about the existence of such inositol-mediated signalling processes in plants, including fungi. In higher plants, inositol is involved in a number of complex metabolic pathways, including formation of phytic acid, an important storage product in seeds (Biswas et al., 1978). In plants, including Saccharomyces cerevisiae and other fungi, inositol is a component of sphingolipids as well as phospholipids (Steiner and Lester, 1972c; Kaul and Lester, 1975,1978; Lester et al., 1978; Hanson and Lester, 1980b). Although phosphatidylinositol (PI) is the major inositol-containing lipid in yeast (Fig. l), inositol-containing sphingolipids represent 4040% of the total inositol-containing lipids in yeast, depending upon growth conditions and strains (Lester et al., 1978). The most abundant inositol-containing sphingolipid in yeast is ceramide (phosphoinositol)2mannose (Cer12P2M; Steiner et al., 1969) and studies of the kinetic labelling and turnover of inositol-containing lipids suggest that the inositol residue in Cer12P2M is derived from PI (Fig. 2; Angus and Lester, 1972). Saccharomyces cerevisiae also contains the di- (PIP) and triphosphorylated (PIP2) forms of PI (Lester and Steiner, 1968) that have been detected in other eukaryotes (Majerus et al., 1988). Turnover of various forms of PI is the source of inositol phosphatides that are thought to play a role as second messengers in a variety of signal-transduction pathways in animal cells (Majerus er al., 1988). Obviously, there is great interest in determining whether similar signal-transduction pathways exist in yeast. As we discuss later, data on this subject are quite preliminary. As a prelude to a discussion of this topic, however, we review the original studies on turnover of inositol-containing lipids in yeast conducted by Lester and his colleagues. Steiner and Lester (1972a) studied metabolism of phosphoinositides (PIPS) in yeast and presented evidence that the phosphates, esterified

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through a monoester linkage to the inositol ring, label and turn over more rapidly than the phosphate which is esterified both to inositol and glycerol (i.e. the phosphate derived from PI). Angus and Lester (1972) showed that PI turns over much more rapidly than other major classes of phospholipids, losing radioactivity with a half-life of about two generations. Almost 75% of 32P present in PI is lost during a four- to six-hour incubation in unlabelled medium, whereas phosphatidylcholine (PC; Fig. 1) continues to acquire

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INOSITOL METABOLISM IN YEASTS

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MICHAEL J. WHITE. JOHN M. LOPES AND SUSAN A. HENRY

label from other cellular constituents for at least four hours under similar conditions and loses little or no label by six hours. Loss of labelled phosphate following PI turnover in yeast coincides with acquisition of label by Cer12P2M, as already discussed, and also with appearance of labelled glycerophosphatidylinositol (GPI) in the growth medium. This last compound is produced at the cell surface by complete deacylation of PI. This extracellular compound reportedly accounts for about half of the labelled inositol and phosphate lost from PI during growth (Angus and Lester, 1975). Under steady-state conditions, extracellular GPI, produced by a growing culture, contains about 25% of the inositol and phosphate found in PI itself. The existence and fate of the major inositolcontaining compounds, GPI and Cer12P2M, must certainly be taken into account in any studies that purport to analyse the role of PI turnover in cellular growth and signalling. Indeed, Angus and Lester (1975) reported that formation of extracellular GPI from PI turnover is influenced by the availability of an energy source. When cells are deprived of glucose, formation of extracellular GPI declines, whereas appearance of free inositol in the growth medium increases. The labelled extracellular inositol that accumulates in the culture containing non-growing cells is also derived from PI turnover (Angus and Lester, 1975). Formation of extracellular GPI has been observed in a number of fungi in addition to Sacch. cerevisiae, including Neurospora spp. (Germanier, 1959) and Schizosaccharomyces pombe (Fernandez et al., 1986). Since much effort is now being focused upon the role of phospholipid breakdown and metabolism in cell-growth control and signalling mechanisms, it would appear that further investigation into the role of GPI formation is required. 11. Biosynthesis of Inositol

Most eukaryotic organisms have the capacity to synthesize inositol 1phosphate (IlP) de novo by conversion of glucose &phosphate (G6P; Fig. 2). Inositol-l-phosphate synthase (IlPS) activity has been detected in mammalian tissues (Eisenberg, 1967; Hasegawa and Eisenberg, 198l), higher plants (Ogunyemi et al., 1978) and a variety of fungi, including Neurospora sp. (Pina et al., 1978) and Sacch. cerevisiae (Culbertson et al., 1976a). Interestingly, however, this enzymic activity is absent from Schiz. pombe (Fernandez et al., 1986), thereby causing this organism to be naturally auxotrophic for inositol. The first step in biosynthesis of inositol and inositol-containing phospholipids is the conversion of G6P to I l P , followed by removal of the phosphate from I1P to yield free inositol (Fig. 2). Free inositol is subsequently

INOSITOL METABOIJSM IN YEASTS

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incorporated directly into phospholipids. Biosynthesis of inositol in a yeast was first demonstrated by Chen and Charalampous (1964a,b, 1965), who analysed crude extracts of Candida utilis. They demonstrated a two-enzyme system for biosynthesis of inositoi: G6P-IlP IlP-+inositol

(1) (2)

The first reaction in this sequence is catalysed by IlPS and the second by I1P phosphatase. Although these workers partially purified and characterized each of these enzymic activities, they were careful to point out that they had presented no evidence that IlPS activity is due to a single enzyme or that a single phosphatase is responsible for the second reaction (Charalampous and Chen, 1966; Chen and Charalampous, l965,1966a,b). The presence of similar enzymic activities in Sacch. cerevisiae was documented by Culbertson et af. (1976a), who also showed that IlPS activity is repressed some 50-fold in Sacch. cerevisiae grown in the presence of inositol concentrations exceeding 50 PM. Synthesis of I1P in vitro is dependent upon NAD+, while NADH inhibits the reaction. However, no net NADH is produced. Inositol 1-phosphate accumulates ir; vitro in the reaction mixture in the absence of magnesium ions, suggesting that the I1P phosphatase is dependent upon these ions. In addition, Culbertson and Henry (1975) isolated mutants of Sacch. cerevisiae that are auxotrophic for inositol, while Culbertson et al. (1976b) showed that these mutants all lack IlPS activity. Inositol-l-phosphate synthase has been purified or partially purified from a number of organisms (Ogunyemi et al., 1978; Pina et al., 1978; Maeda and Eisenberg, 1980), including Sacch. cerevisiae. Donahue and Henry (1981b) purified IlPS from Sacch. cerevisiae some 500-fold to homogeneity. When the purified enzyme is subjected to electrophoresis, a subunit of 62 kDa is detected under denaturing conditions. The native IlPS enzyme is estimated to have a molecular weight of approximately 240,000 by gel-filtration chromatography. Thus, IlPS is believed to be a tetramer of identical 62 kDa subunits. Purified IlPS was used to generate an antibody specific for the 62 kDa subunit. Initially, this antibody was employed to study regulation of expression of the IlPS subunit and to identify the genetic locus that encodes IlPS in Sacch. cerevisiae. In the original collection of inositol auxotrophs isolated by Culbertson and Henry (1975), 52 inositol-requiring mutant isolates representing ten genetic complementation groups (inol-inolO) were reported. Seventy per cent of the mutants were assigned by genetic analysis to the complementation group designated inol. While mutants from all of the complementation groups lack IlPS activity, some of the inol mutants express a 62 kDa protein which is cross-reactive with IlPS antibody (Donahue and Henry, 1981b). This result suggests that some inol mutants

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MICHAEL 1. WHITE, JOHN M. LOPES AND SUSAN A HENRY

express inactive IlPS and that in01 is, therefore, the structural gene for the enzyme. Genetic analysis of the inoZ mutants confirmed that they represent a single genetic locus mapping to chromosome X between URA2 and CDC6 (Donahue and Henry, 1981a). Subsequently, the ZNOZ gene was isolated on an autonomously replicating plasmid by complementation of the inol mutant phenotype (Klig and Henry, 1984). The cloned DNA is capable of correcting the inositol auxotrophy of inoZ mutants, and was shown genetically to be derived from the genomic ZNOZ locus (Klig and Henry, 1984). The DNA sequence of the INOZ gene was obtained, revealing a 553 amino-acid open-reading frame predicted to encode a protein of 62.8 kDa. Consistent with the cytoplasmic location of IlPS activity, the predicted protein lacks obvious membranespanning domains (Dean-Johnson and Henry, 1989). The amino-acid composition and amino-terminal sequence (first eight amino acids) derived from purified IlPS were compared with the protein predicted from the sequence of the open-reading frame of the IN01 gene, confirming that it encodes IlPS (Dean-Johnson and Henry, 1989). 111. Phosphatidylinositol Biosynthesis

Synthesis of PI from inositol and CDP-diacylglycerol (CDP-DG) is catalysed by the membrane-associated enzyme phosphatidylinositol synthase (PIS). This enzyme was purified to homogeneity by Fischl and Carman (1983) and the purification and characteristics of this enzyme were recently reviewed in detail (Carman and Henry, 1989). A mutant, designatedpzs, that exhibited altered PIS activity has also been reported (Nikawa and Yamashita, 1982). Strains bearing the pis mutation are auxotrophic for inositol and can only grow in the presence of high concentrations of inositol. Nikawa and Yamashita (1982) suggested that the pis lesion is due to a mutation in the PIS enzyme that lowers the affinity of the mutant enzyme to roughly 0.5-0.7% of the affinity for inositol exhibited by the wild-type enzyme. A strain harbouring the pis lesion was used to clone a DNA fragment that complements the pis mutation (Nikawa and Yamashita, 1984). The complementing clone also generates eight-fold higher levels of PIS activity when transformed into yeast strains on a high copy-number plasmid. When transformed into the pis strain, the complementing clone restores the affinity of PIS enzyme activity for inositol to the wild-type level. Disruption of the genomic PIS locus was shown to produce a lethal phenotype, confirming that the PIS gene encodes an essential factor (Nikawa et al., 1987a). An open-reading frame of 220 amino-acid residus predicting a protein of

INOSITOL METABOLISM IN YEASTS

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24 kDa was identified as the PZS-coding sequence (Nikawa et al., 1987a). Hydropathy analysis of the PIS DNA sequence revealed two extended hydrophobic regions consistent with a membrane-associated product (Nikawa et al., 1987a). The molecular weight of 24,000 for the gene product of the PZS gene, however, is in disagreement with the 34,000 molecular weight for the PIS subunit of the enzyme purified by Fischl and Carman (1983). This discrepancy could reflect post-translational processing of the 24 kDa protein predicted by the PIS DNA sequence. The sequence identified by Nikawa et al. (1987a) is predicted to have two potential N-linked glycosylation sites. However, there is, as yet, no confirmation that purified PIS protein is modified post-translationally (Carman and Henry, 1989). For these reasons, identification of the PIS gene cloned by Nikawa et al. (1987a) as the structural gene for the subunit of the PIS enzyme purified by Fischl and Carman (1983) must be considered inconclusive at present. Fischl et al. (1986) reconstituted the purified PIS protein into phospholipid vesicles and studied its activity in response to water-soluble precursors and the phospholipid composition of the vesicles. Phosphatidylserine (PS) was found to stimulate PIS activity, suggesting that the phospholipid environment is a factor in regulation of this enzyme. Phosphatidylinositol synthase activity is not, however, regulated in response to the presence of soluble precursors of phospholipid synthesis either in vivo or in vitro (Klig et al., 1985; Fischl etal., 1986). The presence of choline, serine, ethanolamine, CDP-choline, CDP-ethanolamine or glycerol 3-phosphate has no effect upon the reconstituted enzyme in vitro. Furthermore, growth of cells in the presence of inositol, inositol together with choline, or inositol with ethanolamine has no effect on this enzyme activity or on the level of the PIS subunit (Klig et al., 1985; Fischl etal., 1986). To date, the only level on which PIS activity has been shown to be regulated is in response to the phospholipid environment of the membrane (Fischl etal., 1986). Regulation of transcription of the PIS gene cloned by Nikawa et al. (1987a) has not yet been reported, although a 1.2 kbp transcript has been identified. Despite the apparent lack of regulation of PIS activity in response to soluble precursors, there is a rapid increase in the rate of PI biosynthesis when inositol is added to a growing yeast culture (Kelley et al., 1988). This response is too rapid to be due to derepression of enzyme activity by a transcriptional mechanism and it appears to be due largely to preferential utilization of the CDP-DG precursor for PI biosynthesis at the expense of PS biosynthesis. This occurs, at least in part, because phosphatidylserine synthase (PSS), the enzyme that competes directly with PIS for the CDPDG substrate (Fig. 2), is inhibited non-competitively by inositol (Kelley et al., 1988). The rate of PI biosynthesis is also controlled in part by regulation of inositol availability via the reaction catalysed by IlPS. As we will discuss

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INOSITOL METABOLISM IN YEASTS

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CDP-DG synthase (CDP-DGS) is repressed in response to inositol and choline, as is PSS, which competes with PIS for available CDP-DG. Thus, the yeast cell controls PI biosynthesis on multiple levels even though PIS activity is constitutive. In subsequent sections of this review, the multiple levels of regulation that influence or potentially influence PI biosynthesis will be discussed in further detail.

1V. Phosphatidylinositol Kinases It has been postulated that PIPs function as second messengers in membrane signal transduction pathways (Fig. 3). The first step in synthesis of these compounds (Figs 2.and 3) involves phosphorylation of PI to form PIP and PIP2. At least two enzymes are believed to catalyse these reactions, namely PI kinase (PIK) and PIP kinase (PIPK). At present, most of our knowledge of the synthesis of PIPs has come from analysis of multicellular organisms (Inhorn et al., 1987; Majerus et al., 1988). Lester and Steiner (1968) demonstrated that yeast cell membranes include PIPs, and the enzyme activities capable of catalysing synthesis of these compounds were shown to exist in yeast (Wheeler et al., 1972). Wheeler et al. (1972) provided evidence that most of the kinase activity, responsible for incorporation of exogenous [y”P]ATP into PIP2, is located in the plasma-membrane fraction. Purification of PIK from yeast proved cumbersome owing to the general difficulty of isolating membraneassociated enzymes and, in particular, the extreme lability of these enzymes when removed from their native milieu (McKenzie and Carman, 1983; Belunis et al., 1988). Despite these difficulties, Belunis et al. (1988) devised a procedure for purifying PIK from yeast cell membranes. Their strategy resulted in an 8000-fold purification of PIK activity with a 6.3% yield. The PIK activity described by Belunis et al. (1988) is associated with a 35 kDa membrane-associated protein that is converted, upon prolonged storage, to a 30 kDa protein with no loss of specific activity. The product of the reaction catalysed by the purified enzyme was shown to be phosphatidylinositol 4phosphate (PI4P; Belunis et al., 1988). Recently, Auger et al. (1989) demonstrated that yeast cell membranes also contain phosphatidylinositol 3-phosphate (PI3P) and they identified a specific membrane-associated activity that could catalyse synthesis of PI3P from PI. To identify PI3P and to quantitate its steady-state level in vivo, cells were grown in the presence of [3H]inositol and the membrane components were fractionated by highpressure liquid chromatography (HPLC; Auger et al., 1989). The experiments of Auger et al. (1989) suggest that PI3P represents 50% of the total PIP in the membranes of yeast cells. These observations imply that PI kinase

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MICHAEL J WHITE, JOHN M LOPES A N D SUSAN A HENRY

activity in yeast is probably heterogenous and must consist of PI4P and PI3P kinases. Therefore, the enzyme purified by Belunis et al. (1988) is probably only one of several enzymes capable of phosphorylating PI. Indeed, there is also evidence that more than one PI4P kinase exists (G. Carman, personal communication). In support of this, the laboratory of J. Thorner (personal communication) has succeeded in purifying a soluble (not membraneassociated) PI4P kinase that has properties quite different from the enzyme purified by Belunis et al. (1988). Important questions also remain concerning the regulation of PIK. Activity of this enzyme in yeast has been reported to change during batch growth and to be sensitive to intracellular CAMP levels (Holland et al., 1988; Kato et al., 1989). However, the conclusions reached by Holland et al. (1988) and Kato et al. (1989) are in disagreement. Holland et al. (1988) studied regulation of PIK activity as affected by the phase of batch growth and demonstrated that PIK activity is increased two- to 2.5-fold as cells enter the stationary phase of growth. The increase in PIK activity is correlated with a dramatic drop in cAMP levels in stationary-phase cells. To investigate this correlation further, Holland et al. (1988) pre-incubated cellular extracts under assay conditions favouring protein phosphorylation and compared them with extracts assayed under conditions favouring dephosphorylation. Under these in vitro conditions, PIK activity was lowered when CAMPdependent protein phosphorylation was favoured, and increased under conditions favouring dephosphorylation. Kato et al. (1989), however, reported the opposite correlation in experiments employing mutant yeast strains. They measured PIK and PIPK activity in extracts of wild-type yeast cells and compared them with strains harbouring mutations in genes known to affect the CAMP-dependent phosphorylation pathway. In their studies, extracts prepared from strains harbouring rasl, ras2 or cyrl mutations (Fig. 3; Matsumoto et af., 1982) exhibited 3040% less PIK and PIPK activity than wild-type extracts assayed under similar conditions. The two ras mutations result in defects in GTP-binding proteins which stimulate adenylate cyclase, while cyrl produces a defect in adenylate cyclase (Fig. 3). Therefore, these mutations result in lower levels of endogenous CAMP, a situation that presumably results in decreased CAMP-dependent protein phosphorylation . Moreover, addition of exogenous cAMP to cellular extracts produced from a cyrl mutant strain restored PIK and PIPK activities to the elevated levels observed in wild-type extracts. Consistent with these observations, extracts prepared from the bcyl mutant also exhibited PIK and PIPK activity levels double that of wild-type cell extracts. The bcyl mutation is a lesion in the regulatory subunit of CAMP-dependent protein kinase (Fig. 3) that results in elevated CAMP-independent kinase activity. The CAMP-dependent protein

INOSITOL METABOLISM IN YEASTS

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kinase in bcyl mutants is no longer functionally dependent on cAMP due to the lesion in the regulatory subunit. For this reason, a rasl, ras2, bcyl triplemutant strain does not require cAMP for growth, while a rasl, ras2 doublemutant strain is dependent on cAMP for growth. Cell extracts prepared from the triple-mutant strain are similar to extracts from the bcyl mutant strain in that they have double the activities of PIK and PIPK than do comparable wild-type cell extracts. Neither mutant strain shows elevated levels of endogenous CAMP. On the basis of their studies with mutant strains, Kato et al. (1989) reached the conclusion that PIK and PIPK activities are increased under conditions favouring CAMP-dependent protein phosphorylation, while Holland et al. (1988) reached the opposite conclusion. These conflicting conclusions were obtained in experiments using quite different strains, growth conditions and assay conditions. Among other explanations, as already discussed, there may be a heterogeneous population of PI kinases that respond differently to phosphorylation. The discovery in yeast of PI3P is consistent with this possibility. Since both groups of investigators used different assay conditions it is possible that each was in effect looking at a different PI kinase (or kinases). Moreover, it is important to note, as do Holland et al. (1988), that none of these experiments addresses directly the question of regulation of individual PI kinases or their roles in the yeast cell. Cloning of the genes encoding the PI kinases may be necessary to resolve these issues. V. Role of Phosphatidylinositol and Phosphoinositides in Yeast

The essential role(s) of inositol-containing metabolites in fungi became evident with the discovery of a phenomenon known as inositol-less death, first described in Neurospora crussa by Strauss (1958). Inositol-requiring mutants of many species of fungi (Strauss, 1958; Shatkin and Tatum, 1961; Thomas, 1972), including Succh. cerevisiae (Henry etal., 1977), die rapidly if deprived of inositol under conditions that are otherwise growth-supporting. In contrast, mutants auxotrophic for many other compounds, including amino acids, purines or pyrimidines, stop dividing and lose viability comparatively slowly when starved of their requirement (Strauss, 1958; Henry et al., 1977). Under conditions of inositol deprivation, inositolrequiring mutants of Sacch. cerevisiae rapidly cease synthesis of PI (Henry et al., 1977; Becker and Lester, 1977) while macromolecule synthesis continues unimpeded. Synthesis of the inositol-containing sphingolipids is also rapidly affected in inositol-starved cells (Hanson and Lester, 1980a). The synthesis of major components of the cell wall, mannan and glycan, is

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MICHAEL 1. WHITE, JOHN M. LOPES AND SUSAN A HENRY

inhibited immediately following the decline in synthesis of the inositolcontaining sphingolipids and PI (Hanson and Lester, 1980a). Inhibition of synthesis of cell-wall components is a very early consequence of inositol deprivation, and substantially precedes the decline in macromolecule synthesis, Hanson and Lester (1982) reported that activity of the major enzyme catalysing the formation of N-acetylglucosaminylpyrophosphoryldolichol (dol-P2GlcNAc) is diminished in membranes isolated from inositolstarved cells. The enzyme GlcNAc-1P transferase catalyses an initial step in synthesis of N-linked mannans. Activity of this enzyme is decreased by some 70% in membrane preparations derived from cells starved of inositol for three hours. Addition of solubilized PI to such membrane preparations stimulates the enzyme in vitro. Thus, it is believed that inhibition of cell-wall biosynthesis in inositol-starved cells is due to a requirement of the membrane-associated enzyme GlcNAc-1P transferase for PI (Hanson and Lester, 1982). A block in cell-wall biosynthesis during inositol limitation explains much of the phenomenology of altered hyphal growth patterns and colony morphology described in cultures of N . crmsa growing under inositollimited conditions (Shatkin and Tatum, 1961; Hanson, 1980). It also conceivably explains the rapid cessation of cell division observed in inositolstarved cultures of Sacch. cerevisiae. Cessation of cell division during inositol starvation substantially precedes termination of macromolecule synthesis (Henry et al., 1977). However, it appears that overall plasmamembrane biogenesis and expansion are also affected rather early during inositol starvation. Inositol-limited cells, stripped of their walls, metabolize and synthesize macromolecules for several hours (Atkinson et al., 1977). However, such sphaeroplasts, in contrast to inositol-sufficient sphaeroplasts, become osmotically unstable and can be preserved only by gradually raising the content of osmoticum in the suspension. Inositol-deficient sphaeroplasts appear unable to expand in volume beyond a certain point, but they are capable of overall metabolism and macromolecule synthesis even after they become osmotically unstable. The content of dissolved compounds in the cytoplasm appears to rise during inositol limitation through continuing metabolism inside the cell or sphaeroplast which is no longer increasing in volume. The consequent imbalance can be off-set in sphaeroplasts only by raising the content of osmoticum in the culture medium. These results imply that overall expansion of the plasma membrane is affected early during inositol limitation (Atkinson et al., 1977). The mechanism of the apparent inhibition of plasma-membrane growth during inositol deprivation is not at all clear since synthesis of all phospholipids other than PI proceeds unabated during inositol starvation (Becker and Lester, 1977; Henry et al., 1977).

INOSITOL METABOLISM IN YEASTS

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Despite a literature spanning several decades, the mechanism of inositolless death is not understood. It does not appear that lysis or leakage of cell contents is an early event during inositol starvation (Henry et al., 1977).For example, loss of ability to transport and retain glucose, in a glucoseretention assay, is a relatively late event during inositol starvation, concomitant with the first detectable loss in viability (Ulaszewski et al., 1978). Furthermore, inositol-starved cells retain their basic integrity as viewed under the light microscope using vital stains up to the time that viability loss is first detected, and they do not appear to lyse (Henry et al., 1977). However, inositol-deficient cells become quite dense due to continuing metabolism within cells that are no longer dividing or expanding in volume (Henry et al., 1977; Atkinson et al., 1977). This phenomenon has been called “unbalanced growth” (Strauss, 1958; Shatkin and Tatum, 1961; Henry et al., 1977). It may well be that inositol-less death comes about because cessation of synthesis of essential components of the cell wall and plasma membrane is not coupled or co-ordinated with an orderly cessation of overall cellular metabolism. Such an imbalance could lead to catastrophic metabolic failure. Another early study of the role of inositol-containing phospholipids in yeast was the report of Cerbon (1970) of arsenate-adapted yeast cells. From wild-type populations of Sacch. carkbergemis one can isolate colonies that are able to grow in the presence of 10 mM arsenate. These are designated AsAd (arsenate adapted). Cerbon (1970) reported that AsAd cells contain twice as much inositol-containing lipid as their ASS (arsenate sensitive) wild-type counterparts. No difference in the steady-state contents of other phospholipids or fatty-acyl residues was observed. The function of elevated PI contents in promoting resistance to arsenate is not understood. Recently, it has been sugested that PI in the membranes of a number of eukaryotic species may function as an anchor for the covalent attachment of a specific class of glycoproteins (Low and Saltiel, 1988). Conzelmann et al. (1988) reported that a 125 kDa membrane glycoprotein in yeast was anchored in a similar fashion to PI. They demonstrated that the protein contained inositol and fatty-acyl residues by metabolic labelling and showed that the protein could be released from the membrane by a PI-specific phospholipase C. Conzelmann et al. (1988) believe that the glycoprotein anchor in yeast is most likely PI, but they do not rule out the possibility that the anchor could be an inositol-containing sphingolipid. They also found that the temperature-sensitive, secretion-deficient secZ8 yeast mutant could not add the phospholipid anchor to newly synthesized glycoproteins. The secZ8 mutant is blocked in transport of glycoproteins from the endoplasmic reticulum, suggesting that PI anchors are added in the endoplasmic reticulum.

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MICHAEI. J. WHITE, JOHN M. LOPES AND SUSAN A. HENRY

Talwalkar and Lester (1973) presented the first description of PIP and PIP2metabolism in yeast in response to glucose starvation and supplementation. They showed that 32Plabel in PIP and PIP2drops rapidly in comparison with phosphate contained in other lipids, including PI, when growing cells are removed from glucose-containing medium and shifted to buffer. Within one minute after the shift to non-nutrient buffer, over 70% of the 32Plabel was lost from PIP?, and 50% from PIP. After 30 minutes, these compounds contained only 10 and 28%, respectively, of their initial 32P label. In contrast, the 32Pcontent of PI was unaffected after 30 minutes of incubation in buffer. The only compound to show a greater loss in 32Plabel than PIP2 was ATP, which lost 98% of the 32Plabel within one minute. When cells that have been in buffer for 30 minutes were added to fresh glucose-containing growth medium, restoration of ATP, PIP2 and PIP contents followed virtually identical kinetics. All three compounds were restored to their original levels within two minutes. In a related experiment, ATP levels were depleted in vivo in a growing culture by addition of 2-deoxyglucose to the growth medium (Talwalkar and Lester, 1973). Again, a close correlation was observed between the drop in ATP contents in response to growth in the presence of 2-deoxyglucose and the drop in contents of PIP2 and PIP. Thus, it would seem that the contents of these lipids are strongly correlated with cellular ATP levels. Current studies on PI turnover in yeast in response to glucose starvation, growth or cell cycle must certainly take into account these observations of Talwalkar and Lester (1973). The effect of glucose starvation on PIP turnover has also been reported by Kaibuchi et al. (1986). When yeast cells grown in media containing 2% glucose were transferred to a buffer containing 0.02% glucose (starvation conditions), the cells arrested at the Go/GI phase of the cell cycle. Furthermore, when glucose was added to these cultures, the cell-cycle arrest was relieved and 32Pirapidly incorporated, in a time-dependent fashion, into phosphatidic acid (PA), PI, PIP and PIP2. There was little or no incorporation into the other membrane phospholipids under these conditions. In addition, Kaibuchi et af. (1986) noted a rapid appearance of 32Piin inositol4phosphate (14P), inositol 1,4,5-triphosphate (I1,4,5P3) and GPI. These compounds were presumably produced from the PIPs by the action of phospholipases C, A l , and A2 (Figs 2 and 3). The data of Kaibuchi et al. (1986) suggest that addition of glucose to starved cells stimulates turnover of PIPs, rather than synthesis of PIPs. Uno et af. (1988) reported that a monoclonal antibody specific for PIP, caused arrest of cells at the GdGl phase of the cell cycle (for 9&120 minutes) when introduced into yeast cells by electroporation. This observation led them to conclude that PIP2 plays a role in transition through the cell cycle, and that it may conceivably function as a mediator in the response to

INOSITOL METABOLISM IN YEASTS

17

glucose. Starting with a collection of temperature-sensitive mutants, they used electroporation to identify mutants that are super-sensitive to PIP2 antibody (arrested for three hours at the permissive temperature). These mutants were designatedpim forphosphoinositide metabolism (Fig. 3). It is noteworthy that two of the mutants identified in this fashion appear to be defective in PIK (pim2) and PIPK activities (piml; Fig. 3). Moreover, cellcycle arrest in these mutants could be relieved by introducing PIP2 via electroporation. Electroporation of PIP2 into the mutant cells led to a decrease in the number of unbudded cells and allowed one round of cell division to occur. On this basis, Uno et al. (1988) concluded that PIP2 is a rate-limiting factor in the cell-cycle arrest. The only components other than PIP2from the PI pathway that could relieve the cell-cycle arrest of the piml and pim2 mutants were a combination of IP3 and a synthetic form of diacylglycerol (DAG). Both of these compounds are products of phospholipase C-cleaved PIP2 (Fig. 3). Evidence from work on mammalian cells suggests that certain hormones stimulate production of inositol phosphates resulting in efflux of calcium ions from intracellular stores (Fig. 3). Efflux of calcium ions, in turn, is important for progression through the cell cycle. Kaibuchi et al. (1986) showed that efflux of calcium ions occurred in yeast concomitant with 32Pi incorporation into inositol, and that these two events followed similar kinetics. The general turnover of PIPS and efflux of calcium ions were observed in response to mannose and fructose as well as glucose (Kaibuchi ef al., 1986), but non-metabolizable forms of glucose, such as 2-deoxyglucose or 6-deoxyglucose, did not produce this effect (Talwalkar and Lester, 1973; Kaibuchi et al. , 1986). Ergosterol reportedly also stimulates PIP turnover and cell proliferation under certain conditions in an ergosterol-requiring strain (Dahl and Dahl, 1985). A yeast strain that is auxotrophic for sterols and unsaturated fatty acids, namely GL7, arrested at the GdG, phase of the cell cycle when starved for ergosterol. Whereas ergosterol is a natural yeast sterol, the GL7 strain grew at a slow rate (generation time, six hours) in media containing cholesterol. If, however, cells growing in cholesterol were shifted to media containing ergosterol (shift-up), the growth rate doubled after a lag of three hours. Dahl and Dahl (1985) quantitated the relative proportions of membrane lipids during the three-hour period following ergosterol shift-up, and noted an initial increase in 32P labelling of PI, PIP and PIP2, with the most significant increase in PIPz labelling. Following the initial burst of labelling, the amount of label in each of these lipids decreased at a rate faster than the turnover observed in cultures not shifted to ergosterol-containing media. Again, the most dramatic turnover was observed for PIP2. Shifting the cells to ergosterol-containing medium increased the growth rate and

18

MICHAEL J . WHITE, JOHN M. LOPES AND SUSAN A HENRY

this, in turn, was correlated with an increased rate of turnover of the PIPS. More recently, Dahl et af. (1987) showed that ergosterol can stimulate PIK activity. Another kinase, antigenically similar to the Rous sarcoma virus pp60"-src,is also reportedly stimulated by ergosterol at concentrations in the range of mammalian hormones (i.e. approximately 1nM). This concentration of sterol cannot, however, relieve the GJG, cell-cycle arrest of the GL7 strain cultured in the absence of ergosterol. These observations led Dahl et al. (1987) to suggest that, in yeast, ergosterol may play a role as a regulator of enzyme activity in addition to its structural role in membranes as a bulk lipid. VI. Role of Inositol in Regulation of Phospholipid Biosynthesis Inositol has been shown to play a central role in regulation of many enzymes of phospholipid synthesis (for a review, see Carman and Henry, 1989). Specifically, IlPS, a cytoplasmic enzyme, and the entire set of genes encoding the membrane-associated enzymes leading to synthesis of PC via methylation of phosphatidylethanolamine (PE; Figs 1 and 2) are repressed to different degrees when inositol is present in the growth medium. Table 1 presents a summary of these data. An interesting feature of this regulation is the concerted effect that inositol and choline exert if they are present simultaneously. With all of the coregulated enzymes, choline has little or no repressive effect if it is present by itself in the growth medium. However, if choline is added to growth medium already containing inositol, the level of repression is greater for many of the enzymes than the level of repression observed when only inositol is present. The physiological purpose of this regulation is not known, but it has been speculated that it plays a role in insuring that the total charge balance of plasma-membrane phospholipids is maintained within certain parameters (Henry et al., 1984; Carman and Henry, 1989). In this regard, it is interesting that PI is a major negatively charged lipid in the yeast plasma membrane, while PC is the major zwitterionic species (Fig. 1). The regulation appears to function to insure that the balance of these two classes of phospholipids is maintained under a variety of growth conditions. Similar regulation of phospholipid charge has been reported in N . c r a m (Hubbard and Brody, 1975). For many of the coregulated enzymes, regulation in response to soluble phospholipid precursors has been studied only at the level of enzyme activity in crude extracts. However, with IlPS and PSS, regulation has also been studied at the level of expression of the enzyme subunit using Western-blot procedures or immunoprecipitation techniques (Donahue and Henry, 1981b; Poole et al., 1986; Homann et a f . , 1987a). In addition, since the

TABLE 1.

Regulation of phospholipid biosynthetic enzymes

Per cent activity in wild-type cells Enzyme

Medium supplement": None Choline Inositol Choline and Inositol

PIS IlPS*

100 100

100 100

100 10

100

PAP CDP-DGS

100 100

100 100

200 70

200 40

PGPS PSS'

100 100

100 100

2545 60

20-45 25

PSD

100

100

40

20

PMTS

100

100

60

10-20

3

Source

Fischl et al. (1986) Culbertson et al. (1976b) Donahue and Henry (1981b) Hirsch and Henry (1986) Morlock et al. (1988) Homann et al. (1985) Klig et al. (1988a) Greenberg et al. (1988) Klig et al. (1985) Poole et al. (1986) Bailis et al. (1987) Homann et al. (1987a) E. Lamping and S. Kohlwein (personal communication) Yamashita et al. (1982) G. Carman (personal communication)

Structural genes encoding for enzymes

Cloned by

PIS IN01

Nikawa and Yamashita (1984) Klig and Henry (1984)

NA NA

NA NA

NA CHOI

NA Letts et al. (1983) Kiyono et al. (1 987) Nikawa et al. (1987a)

NA

NA

CH02 (PEMI) 0PI3 ( P E M 2 )

Kodaki and Yamashita (1987) Summers et al. (1988) McGraw and Henry (1989)

"Medium supplements: choline, 1 mM; inositol, 75 PM. bRelative values determined at the level of IN01 mRNA abundance. 'Relative values determined by enzyme activity, Western-blot analysis of PSS subunit abundance and abundance of C H O I mRNA. NA, data not available. All other values in the table are expressed as percentage of enzyme activity relative to derepressed values (no supplement).

20

MICHAEL J. WHITE, JOHN M. LOPES AND SUSAN A . HENRY

structural genes encoding IlPS and PSS have been cloned, it has been possible to study regulation using the cloned genes as probes. In this fashion, it has been shown that regulation of IlPS (Hirsch and Henry, 1986) and PSS (Bailis et al., 1987) in response to inositol and choline occurs at the level of transcript abundance. Since all of the coregulated enzymes exhibit a similar pattern of regulation, and since all respond to a common set of regulatory genes as will be discussed subsequently, it is believed that this regulatory control is mediated through a common set of transcription factors. In the remaining section of this review, we discuss details of this regulation. As already discussed, PIS, the enzyme directly responsible for synthesis of PI from CDP-DG and free inositol, is not one of the enzymes under regulation in response to inositol and choline. Phosphatidylinositol synthase activity is constitutive. However, as we have already stated, PI biosynthesis is regulated indirectly via this mechanism since the rate of PI biosynthesis is influenced by the availability of the precursors, namely inositol and CDPDG (Kelley et al., 1988). A. REGULATION OF INOSITOL 1-PHOSPHATE BIOSYNTHESIS

Inositol-l-phosphate synthase is the most highly regulated of the enzymes that are repressed in response to inositol (Table 1). Growth of yeast in media supplemented with 50 p~ inositol lowers the activity of this enzyme to 2% of that observed in extracts from cells grown in the absence of inositol (Culbertson et al., 1976a). This decrease in IlPS activity correlates with a drop in the level of immunoprecipitable IlPS subunit (Donahue and Henry, 1981b) . Inositol-l-phosphate synthase is encoded by the I N 0 1 gene, and it has been shown that the ZNOZ clone hybridizes to a 1.8 kbp poly(Af) RNA (Hirsch and Henry, 1986). Accumulation of this transcript is affected by the presence of inositol and choline in the growth medium. In particular, a decrease in the level of this transcript to 8% of the derepressed level is observed when cells are grown in the presence of 75 ,UMinositol. In addition, 1 mM choline by itself has no effect on transcription of ZNOZ, but growth of c e k in the presence of a combination of 1 r n chdine ~ and 75 FM inositol results in a further decrease in the level of the ZNOZ transcript to 3% of the derepressed level (Hirsch and Henry, 1986). The level of ZNOZ transcript is also influenced by mutations in regulatory genes, a topic that is discussed in a subsequent section of this review. Analysis of the INOZ promoter cis-acting regulatory sequences is currently in progress. Towards that end, a fusion of the I N 0 2 promoter to the lucZ gene has been constructed and integrated into the yeast genome. Transcription of this fusion gene has been shown to respond to growth in the

INOSITOL METABOLISM IN YEASTS

21

presence of the phospholipid precursors (Hirsch, 1987). The amount of the ZNOZ’facZ fusion RNA is lowered to 10% of the derepressed level in cells grown in the presence of inositol and 5% of the derepressed level in cells grown in the presence of inositol and choline. The relative level of expression of the INOl ‘lacZ fusion RNA under different growth conditions is similar to the relative level of expression of native I N 0 2 RNA under similar conditions (Hirsch and Henry, 1986). Thus, sequences responsible for transcriptional regulation of the INOl gene are contained in the fusion gene. A selective deletion analysis of the I N 0 2 promoter region suggests that at least two elements in the promoter are required for repression since omission of these regions results in constitutive derepressed expression of the ZNOZ’lacZ fusion. The function of these sites in the promoter and their interaction with identified regulatory factors are discussed in depth in a subsequent section of this review. B. REGULATION OF PHOSPHATIDIC-ACID PHOSPHATASE

The enzyme phosphatidic-acid phosphatase (PAP) catalyses dephosphorylation of PA to yield D A G (Fig. 2), which in turn is used to synthesize triacylglycerols (TAG) or PC via a salvage pathway (Carman and Henry, 1989). This enzyme activity, therefore, may play a key role in determining the flow of substrate between two major branches of the phospholipid biosynthetic pathway (Fig. 2). In particular, flow of substrate in the direction of DAG decreases the amount of substrate flowing in the direction of CDPDG, the immediate precursor of PI. As we have already discussed, the availability of CDP-DG may play an important role in control of PI biosynthesis (Kelley et al., 1988). Partial purification of PAP revealed that this enzyme is localized in soluble as well as membrane fractions (Hosaka and Yamashita, 1984). However, it is not yet known whether the two forms of the enzyme are encoded by a single gene or several genes. As yeast cultures enter the stationary phase of growth, a dramatic ten-fold increase in the level of TAG (Fig. 2) is observed while the content of phospholipids is virtually unchanged (Hosaka and Yamashita, 1984). Levels of TAG increase during sporulation and during growth in phosphate- and inositol-deficient media. Since PAP is involved in synthesis of this abundant class of lipids, regulation of this enzyme may play an important role in controlling levels of TAG. Analysis of soluble and membrane fractions obtained from yeast cultures taken at different stages of growth revealed a significant increase in PAP activity (1.7-fold) as cultures entered the stationary phase of growth (Hosaka and Yamashita, 1984). However, these experiments were performed using cells grown in the presence of inositol, and it was later shown

22

MICHAEL J. WHITE. JOHN M LOPES AND SUSAN A. HENRY

by Morlock et af. (1988) that growth in the presence of inositol results in a two-fold increase in PAP activity. In the presence of inositol, a four-fold increase in PAP activity was observed in cultures grown to the stationary phase as compared with those growing exponentially in the absence of inositol (Morlock et al., 1988). This enzyme is the only one so far identified that is induced by inositol (Table 1). All other enzymes reported in Table 1, with the exception of PIS, are repressed in the presence of inositol. This observation may be significant in terms of overall regulation of phospholipid metabolism, since the reaction catalysed by PAP competes with the reaction catalysed by CDP-DGS for PA. Inositol represses CDP-DGS, as does a combination of inositol and choline (Table 1).Thus, when both inositol and choline are present, DAG production may be favoured at the expense of CDP-DG production. C. REGULATION OF CDP-DIACYLGLYCEROL SYNTHASE

As discussed in the preceding section, the enzyme CDP-DGS catalyses activation of PA to CDP-DG (Fig. 2). The branchpoint precursor for the synthesis of PI and PS is CDP-DG (Fig. 2). Phosphatidylserine, in turn, serves as a precursor for synthesis of PE and, ultimately, PC. Kelley and Carman (1987) purified CDP-DGS (2400-fold) from Sacch. cerevisiue to homogeneity. Details of the purification and characteristics of the enzyme were recently reviewed (Carman and Henry, 1989). When fractionated on a non-denaturing gel, CDP-DGS activity was predominantly associated with a high molecular-weight complex (114 OOO) that is apparently composed of two smaller subunits of 54 and 56 kDa. Neither of the separated subunits yielded any CDP-DGS activity (Kelley and Carman, 1987). However, antibodies raised in response to the native enzyme or in response to either of the two subunits precipitated a protein with CDP-DGS activity that fractionated into two subunits of 54 and 56 kDa (Kelley and Carman, 1987). The two subunits of different molecular weight were observed even if several protease inhibitors were included during isolation and purification, suggesting that the smaller subunit does not result from proteolytic cleavage of the larger subunit. However, both subunits have remarkably similar amino-acid compositions, and additional information will be required to determine if they are encoded by the same gene or two different genes. One of the enzymes regulated in response to the presence of water-soluble phospholipid precursors, including inositol, in the growth medium is CDPDGS (Table 1;Homann et al., 1985;Klig et al., 1988a). Regulation of CDPDGS activity may be crucial to control of PI biosynthesis since the availability of CDP-DG and inositol precursors appears to control the rate of PI biosynthesis (Kelley et af., 1988). When ethanolamine or choline was

INOSITOL METABOLISM IN YEASTS

23

added to the growth medium, there was n o effect on CDP-DGS activity. However, addition of inositol to the growth medium resulted in a 30% decrease in activity of this enzyme. Moreover, if inositol was present in the growth medium in combination with either ethanolamine or choline, a 60% decrease in CDP-DGS activity was observed (Table 1). The presence of serine also caused decreased CDP-DGS activity if it was present in the growth medium in combination with inositol. Serine alone, however, had no effect (Klig et af., 1988a). Thus, the pattern of regulation of CDP-DGS is similar to that observed for all of the coregulated enzymes listed in Table 1. However, CDP-DGS exhibits the smallest repression ratio of the entire set of coregulated activities. Antibody specific to CDP-DGS subunit has been used to study expression of this enzyme under different growth conditions. A direct correlation between expression of the enzyme subunit and activity of the enzyme was observed under all conditions (Klig et af., 1988a). As with a number of phospholipid biosynthetic enzymes, CDP-DGS activity is also regulated in response to the growth phase of the culture (Homann etal., 1987b). As cultures grown in the absence of inositol entered the stationary phase of growth, a 60% decrease in CDP-DGS activity was observed. A decrease in CDP-DGS activity was not observed in cells from stationary-phase cultures grown in the presence of inositol and choline, presumably because such cultures were already repressed to the maximum extent by the presence of phospholipid precursors. While the structural gene (or genes) encoding CDP-DGS in yeast has not been identified, a mutant (cdgl)with decreased CDP-DGS activity has been isolated (Klig et af.,1988a). Extracts from strains bearing the cdgl mutation exhibited only 25% of the CDP-DGS activity of wild-type extracts (Klig et al., 1988a). The enzyme partially purified from the cdg2 mutant is identical by several biochemical criteria to the enzyme purified from the wild-type strain. The cdg2 mutant has a very pleiotropic phenotype including overproduction of inositol (Opi- phenotype, to be discussed subsequently) and constitutive expression of IlPS, PSS and other enzymes of phospholipid synthesis. At present, the nature of the CDGl gene product is unknown. D. REGULATION OF PHOSPHATIDYLGLYCEROL-PHOSPHATE SYNTHASE

Phosphatidylglycerol-phosphate synthase (PGPS) is an enzyme located primarily in the mitochondria1 membrane of the yeast cell (Kuchler et af., 1986). This enzyme is responsible for catalysing the first in a series of reactions that lead to synthesis of cardiolipin, a lipid primarily associated with mitochondria. Greenberg etaf. (1988) demonstrated that PGPS activity is regulated in response to inositol. The level of enzyme activity in cells grown in the presence of inositol was 45-25'/0 of the fully derepressed level

24

MICHAEL 1. WHITE. JOHN M. LOPES AND SUSAN A. HENRY

observed when precursors were present in the growth medium (Table 1). Addition of choline to medium containing inositol had little additional effect, and choline by itself had no effect on the activity of PGPS. E. REGULATION OF PHOSPHATIDYLSERINE SYNTHASE

In yeast, the membrane-associated enzyme PSS catalyses synthesis of PS from serine and CDP-DG (Fig. 2). Regulation of this reaction plays a particularly important role in controlling PI biosynthesis since it competes directly with PI biosynthesis for available CDP-DG (Fig. 2). As mentioned previously, Kelley et al. (1988) have shown that inositol is a non-competitive inhibitor of PSS activity in vitro.Thus, the presence of inositol inhibits PSS activity, allowing a greater proportion of the common substrate CDP-DG to be utilized for PI biosynthesis. Bae-Lee and Carman (1984) described a purification strategy for PSS using yeast cells, and reported that the subunit of the purified enzyme has a mass of 23 kDa. Other investigators have reported detection of a 30 kDa subunit as well as a 23 kDa subunit (Kiyono et al., 1987; Kohlwein et al., 1988). Hromy and Carman (1986) reconstituted PSS into synthetic vesicles and studied the effect of phospholipid composition on enzyme activity. Phosphatidylserine synthase, like PIS, is influenced by the phospholipid environment. Increasing the ratio of PI to PS in membrane vesicles led to a decrease in PSS activity in vitro (Hromy and Carman, 1986). More recently, it has been shown that PSS is phosphorylated in vivo by a CAMP-dependent protein kinase and that this phosphorylation can be reproduced in vitro (Kinney and Carman, 1988). Phosphorylation of PSS results in a 60-70% decrease in its activity in vitro. Moreover, conditions that lower levels of cAMP in cells, such as growth into stationary phase, decrease the level of phosphorylation of PSS in vivo. A PSS-specific antibody precipitated a phosphorylated version of the 23 kDa subunit of PSS from extracts of cells labelled in vivo with 32Pi.However, in cells grown into stationary phase, there was no detectable phosphorylation of the PSS subunit. Most recently, Kinney et al. (1990) explored the relationship between cellular cAMP levels, PSS phosphorylation and phospholipid synthesis in vzvo, using a cyrl mutant of Succh. cerevisiue (Fig. 3) which is defective in adenylate cyclase (Matsumoto et al., 1982). This mutant lacks CAMPdependent protein-kinase activity when it is grown in the absence of exogenous CAMP, and it arrests in the G, phase of the cell cycle. Cellular levels of cAMP can be controlled in this mutant by regulating concentrations of exogenous cAMP (Matsumoto et al., 1982). Kinney et ul. (1990) grew the cyrl strain in the presence of CAMP and subsequently transferred these cells to medium lacking CAMP.In the absence of CAMP, the rate of PS synthesis

INOSITOL METABOLISM IN YEASTS

25

increased at the expense of PI synthesis, while further addition of cAMP resulted in increased PI biosynthesis at the expense of PS (Kinney et al., 1990). Kinney et al. (1990) also examined activities of a number of phospholipid biosynthetic enzymes in extracts of the cyrl cells, grown in the presence or absence of CAMP. Cells grown in the presence of cAMP had decreased PSS activity compared with cells grown in the absence of CAMP. Cells grown in the presence of cAMP had PSS activity levels comparable to wild-type cells. The decreased PSS activity of the cyrl cells grown in the presence of cAMP correlated with an observed decreased rate of PS biosynthesis. Phosphatidylinositol synthase activity of cyrl mutant cells, on the other hand, was not influenced by cAMP supplementation. These results led Kinney et al. (1990) to suggest that the increase in PI biosynthesis observed in cyrl cells grown in the presence of cAMP is not due to an alteration in PIS activity, but rather to a decreased competition from PS biosynthesis for the common precursor, CDP-DG, due to the down regulation of PSS by phosphorylation. These data provide further support for the contention of Kelley et al. (1988) that regulation of PSS activity is a major mechanism for controlling flow of substrate into the competing reaction catalysed by PIS. Thus, it appears that PI biosynthesis is controlled to a major extent by regulating PS biosynthesis. These results are also entirely consistent with earlier studies of Kinney and Carman (1988) showing that PSS phosphorylation is decreased in vivo as cells enter the stationary phase of growth. Phosphatidylserine synthase is also among the enzymes repressed in response to inositol and choline (Table 1). Extracts prepared from cells grown in the presence of 50 p~ inositol contained only about 60% of the PSS activity detected in extracts from cells grown in the absence of inositol (Poole et al., 1986; Homann et al., 1987a). However, the presence in the growth medium of inositol in combination with choline or other phospholipid precursors, such as ethanolamine or L-serine, resulted in a further decrease of PSS activity to about 20-25% of the fully derepressed level. D-Serine, glycine and cysteine, in combination with inositol, also produced a further decrease in PSS activity to about 20-25% of the derepressed level (Homann et al., 1987a; Poole et al., 1986). None of these compounds has any effect in the absence of inositol. Repression of PSS is not due to a direct action of any of these compounds on the enzyme since their addition to crude extracts did not alter PSS activity. The 23 kDa PSS subunit was studied using Western-blot analysis, revealing a perfect correlation between enzyme activity and quantity of the subunit present in crude extracts of cells grown in the presence of different combinations of precursors. Mutants (chol) defective in PSS activity were identified as auxotrophs unable to grow on media lacking choline or ethanolamine (Lindegren et al.,

26

MlCHAEL J. WHITE, JOHN M. LOPES AND SUSAN A . HENRY

1965; Atkinson et al., 1980b; KovaE et al., 1980; Nikawa and Yamashita, 1981; Letts and Dawes, 1983; Letts and Henry, 1985). The chol mutants are able to grow when supplied with ethanolamine or choline because PE and PC can be synthesized via a salvage pathway (Fig. 2), first described by Kennedy and Weiss (1956), in which CDP-ethanolamine or CDP-choline reacts with DAG to form PE or PC, thus bypassing PS as an intermediate (Atkinson et al., 1980a; KovBt et al., 1980; Letts and Henry, 1985). Virtually no PSS activity was detected in chol strains, whereas the level of PS decarboxylase activity (Atkinson et al., 1980a; KovBE et al., 1980) and phospholipid methyltransferase activity was normal in these strains (Letts and Henry, 1985; Nikawa and Yamashita, 1981). The chol mutants possess membranes virtually devoid of PS, leading to the conclusion that Sacch. cerevisiae can survive and grow when PS is completely absent from cellular membranes (Atkinson et al., 1980a,b). Letts et al. (1983) reported cloning the CHOI gene by complementation of the choline auxotrophy of a chol mutant. Similar clones were also obtained by Nikawa et al. (1987b) and Kiyono et al. (1987). The CHOl gene has also been referred to in the literature as PSS (Nikawa et al., 1987b). Strains bearing a disruption of the genomic CHOl locus [generated by deletion of part of the CHOl sequence (Kiyono et al., 1987), by insertion of a TRPl gene (Bailis et al., 1987) or a LEU2 gene (Hikiji et al., 1988)l are auxotrophic for choline and ethanolamine, lack PSS activity, and do not contain detectable amounts of PS in their membranes. Transformation of a chol mutant strain with any one of the CHOl-containing plasmids resulted in four- to 12-fold higher levels of PSS activity (Letts et al., 1983; Nikawa et al., 1987b; Kiyono et al., 1987), presumably due to the elevated copy number of the CHOl-containing plasmids. Sequence analysis of the CHOl clones revealed an open-reading frame of 276 amino-acid residues corresponding to a 30 kDa protein (Nikawa et al., 1987b; Kiyono et al., 1987). The translation start codon (either of two closely spaced ATG codons) for the CHOl gene was established by comparing the deduced primary aminoacid sequence to the 14 amino-terminal residues of PSS (Kiyono et al., 1987). Biochemical evidence suggests that PSS is translated as a 30 kDa protein that is subsequently cleaved by proteolysis to a 23 kDa protein (Kiyono et al., 1987). These results explain the detection of both 23 and 30 kDa versions of the PSS subunit (Kiyono et al., 1987; Kohlwein et al., 1988). While the role of each form of PSS in vivo remains to be clarified, it is quite clear that there is only a single structural gene (CHO1) that encodes both forms of the enzyme (Kohlwein et al., 1988). Bailis et al. (1987) demonstrated a nearly perfect correlation between relative steady-state levels of the CHOl transcript and the previously reported PSS enzyme levels. This observation is consistent with transcriptional

INOSITOL METABOLISM IN YEASTS

27

regulation of the CHOl gene in response to the soluble precursors. Most recently, a CHOl'lucZ fusion gene has been employed to map the cis-acting elements that compose the CHOl promoter (Bailis, 1988; A. Bailis, personal communication). To that end, several deletions in the region flanking the CHOZ 'lucZ fusion gene have been constructed (A. Bailis, personal communication). This analysis suggests that all of the cis-regulatory elements are contained within 300 bp of DNA immediately flanking the CHOl gene. F. REGULATION OF PHOSPHATIDYLSERINE DECARBOXYLASE

Yeast synthesizes PE de novo by decarboxylation of PS (Carson et ul., 1982). The enzyme that catalyses this reaction is phosphatidylserine decarboxylase (PSD) which is localized in the inner mitochondria1 membrane of yeast cells (Kuchler et al., 1986). Carson et al. (1984) described solubilization of decarboxylase activity from yeast cell membranes and determined several biochemical features of this enzyme activity. Similar to the other enzymes listed in Table 1 , PSD activity is regulated in response to the presence of soluble phospholipid precursors in the growth medium. Initial studies on this enzyme were conducted in medium containing inositol and other compounds (Carson et ul., 1984). While the presence of ethanolamine had no effect on PSD activity, monomethylethanolamine (MME), dimethylethanolamine (DME) or choline added to media containing inositol caused repression of PSD activity. More recently, E. Lamping and S.D. Kohlwein (personal communication) reinvestigated regulation of PSD. They have found that the presence of inositol in the growth medium resulted in a decrease in PSD activity to 38% of the level that was observed in cells grown in the absence of precursors. Addition of ethanolamine or choline to the growth medium in combination with inositol resulted in a further lowering of PSD activity to 21% of the wild-type derepressed level (Table 1).Thus, regulation of PSD is quite similar to regulation of PSS, the enzyme that immediately precedes it on the pathway (Fig. 2). G. REGULATION OF PHOSPHOLIPID METHYLTRANSFERASES

In yeast, as in other eukaryotes, de n o w synthesis of PC involves three sequential methylations of PE with S-adenosylmethionine (SAM) as the methyl donor (Waechter and Lester, 1973; for a review, see Carman and Henry, 1989). There is now a significant body of evidence suggesting that these reactions are catalysed by two membrane-associated enzymes, one of which converts PE to phosphatidylmonomethylethanolamine (PMME) and a second that sequentially converts PMME to phosphatidyldimethylethanolamine (PDME) and PC. Because the reactions catalysed by these

28

MICHAEL J. WHITE, JOHN M LOPES AND SUSAN A HENRY

enzymes share similar features, and since the two activities have yet to be purified to homogeneity, they will be discussed together and denoted collectively as phospholipid methyltransferases (PMTs). Despite the difficulty in purifying the two enzyme activities, it has been possible to isolate mutant strains that harbour genetic lesions defining these two functions (Yamashita and Oshima, 1980; Greenbergetal., 1982b, 1983; Yamashita et al., 1982; Summers et al., 1988). Strains harbouring the mutations designated as peml (Yamashita and Oshima, 1980) or ch02 (Summers et al., 1988) are defective in the first of the two PMT activities. Strains harbouring the chu2 mutations accumulate elevated levels of PE and have severely decreased levels of PC when grown on minimal medium. Interestingly, strains bearing the ch02 mutations are not choline or MME auxotrophs (Summers et al., 1988) while strains bearing the peml mutation (which are biochemicallyindistinguishable from the cho2 strains) reportedly require supplementation (Yamashita et al., 1982). It seems likely, however, that the peml and ch02 mutant strains define a single genetic locus that encodes the first PMT. By the same criteria, mutations designated as opi3 (Greenberg et al., 1982b, 1983) and pem2 (Yamashita et al., 1982) most likely define the second PMT. The original opi3 mutant was identified from a collection of mutants that excrete inositol (Greenberg el al., 1982b, 1983), whereas pem2 mutants are described as strict choline-requiring auxotrophs (Yamashita el al., 1982). The opi3 mutants clearly are not strict cholinerequiring auxotrophs (Greenberg et al., 1983; McGraw and Henry, 1989) since they grow in the absence of supplements, despite having barely detectable levels of PC in their membranes. Despite the difference in the selection schemes employed for isolation of the pem2 and opi3 mutants, both types of mutant accumulate elevated levels of PMME, and exhibit barely detectable levels of PC. A detailed genetic analysis of the cho2 and opi3 mutations has demonstrated that they are recessive and unlinked to each other (Greenberg et al., 1983; Summers et al., 1988; McGraw and Henry, 1989). In further support of the similarity between thepeml and cho2 mutations, clones that complement every feature of each defect have been isolated (PEMI and C H 0 2 , respectively) and possess identical restriction maps (Kodaki and Yamashita, 1987; Summers et al., 1988). The C H 0 2 clone was isolated by complementation of the choline requirement of a ch02 cdgl double mutant (Summers et al., 1988) and was shown to correct every defect associated with the ch02 mutation. The C H 0 2 clone, however, lacks the ability to complement the opi3 genetic lesion. The cloned C H 0 2 gene was used to disrupt its cognate genomic copy by insertion of the LEU2 gene, and the null allele was found to be biochemically and genetically indistinguishable from previously isolated ch02 alleles. In particular, strains bearing the

INOSITOL METABOLISM IN YEASTS

29

ch02 null allele are not choline-requiring auxotrophs (Summers etal., 1988). Kodaki and Yamashita (1987) cloned the PEMl gene by complementation of the choline requirement associated with the peml mutation, and employed the cloned gene to generate a null mutant. The null mutant was biochemically indistinguishable from the previously isolated peml mutations (Kodaki and Yamashita, 1989). They sequenced the PEMZ gene and reported an 869 amino-acid residue open-reading frame predicting a protein of 101 kDa. Both the C H 0 2 and the PEMl clones identify a poly(A+) transcript of approximately 3 kbp in RNA isolated from wild-type strains or ch02 mutants (Kodaki and Yamashita, 1987; Summers et al., 1988). This transcript is completely absent from strains bearing the ch02 null allele (Summers et al., 1988). Clones complementing the genetic defects in the pem2 and opi3 mutants have also been isolated (Kodaki and Yamashita, 1987; McGraw and Henry, 1989). A comparison of the restriction maps of the cloned OPZ3 and PEM2 genes suggests that they are identical (McGraw and Henry, 1989). The PEM2 and OPZ3 clones were obtained not only by complementation of the pem2 and opi3 mutations, respectively, but, unexpectedly, also by complementation of the p e m l and cho2 mutations (Kodaki and Yamashita, 1987; Summers et al., 1988). These PEM2 and OPZ3 clones correct every biochemical defect associated with mutations in the second PMT and, in addition, can relieve some of the defects inherent in strains carrying mutations defining the first PMT. The cloned OPZ3 gene was employed to create a null allele of its cognate genomic copy. The null allele was biochemically and genetically indistinguishable from the various opi3 alleles (McGraw and Henry, 1989) and is not auxotrophic for choline. Similarly, the cloned PEM2 gene has been used to construct a null allele that is biochemically indistinguishable from the pem2 lesion (Kodaki and Yamashita, 1989). Sequence analysis of the PEM2 gene revealed an openreading frame of 206 amino-acid residues with a mass of 23 kDa (Kodaki and Yamashita, 1987). In further confirmation of the identity of the PEM2 and OPZ3 clones, a 0.9 kbp transcript was detected by Northern-blot analysis of poly(A+)-selected RNA using either clone as a probe (Kodaki and Yamashita, 1987; McGraw and Henry, 1989). As with other phospholipid biosynthetic enzymes listed in Table 1, PMTs are also regulated in response to the presence of soluble precursors (Yamashita et al., 1982; G. Carman, personal communication). While growth in the presence of inositol or choline causes a modest decrease in PMT activity (a 36 and 14% decrease, respectively), the two precursors in combination caused a 79% decrease (Yamashita et al., 1982). Recent experiments suggest that this reduction is controlled, at least in part, at the level of transcription (S. Toutenhoofd and T. Gill, personal communication).

30

MICHAEL J. WHITE, JOHN M. LOPES AND SUSAN A. HENRY

Growth of cells in the presence of the individual precursors appears to elevate transcription of the C H 0 2 and OF13 genes, whereas combinations of these compounds decrease transcription. In addition, Homann et al. (1987b) also demonstrated that PMT activity is affected by the growth phase in which a culture is harvested. Phospholipid methyltransferase activity is decreased by 60-70% as cells enter the stationary phase of growth. VII. Interconnection between Phosphatidylcholine Biosynthesis and Regulation of Phospholipid Biosynthesis by Inositol

Analysis of yeast mutants defective in PC biosynthesis produced the startling observation that regulation of IlPS in response to inositol is entirely dependent upon ongoing PC biosynthesis. As discussed in the preceding sections, there exist three well-defined classes of mutants of Sacch. cerevisiae that have defects in structural genes encoding enzymes directly involved in the de novo synthesis of PC, namely chol, cho2lpeml and opi3I pem2. The chol mutants, as described previously, have defects in PSS, while the cho2lpeml and opi3lpem2 lesions define the two PMTs. With the CHOl locus, the evidence identifying it as the structural gene for PSS is unambiguous and definitive. The DNA sequence of the CHOl gene predicts an amino-acid sequence for its gene product that matches the experimentally determined N-terminus of the isolated protein (Kiyono et al., 1987). Furthermore, antibody produced in response to the product of the cloned gene cross-reacts with the PSS subunit (Kohlwein et al., 1988).As regards to the PMTs, the gene products have not been studied at the protein level. Thus, the evidence supporting identification of the CHO2IPEMl and OP13l PEM2 genes as structural genes encoding the phospholipid methyltransferases is compelling, but not entirely definitive. Mutant yeast strains bearing lesions in each of these three genes have a curious secondary phenotype. They are conditional Opi- mutants, that is to say they overproduce inositol and excrete it into the growth medium under certain growth conditions. This observation led to the discovery that the yeast cell is incapable of regulating IlPS expression in response to exogenous inositol if PC synthesis is blocked. When choline or ethanolamine is supplied to chol mutants, PC biosynthesis occurs. Under these conditions, chol strains have no inositol overproduction (Opi-) phenotype. Furthermore, under conditions permitting PC biosynthesis (i.e. when ethanolamine or choline is present), IlPS expression is regulated normally in response to inositol (Letts and Henry, 1985). When deprived of choline and ethanolamine, chol cells cannot synthesize PC and they eventually stop growing, but d o not die. When choline (or ethanolamine) is removed from the growth

INOSITOL METABOLISM IN YEASTS

31

media, these cells derepress IlPS whether or not inositol is present. Furthermore, choZ cells excrete inositol into the growth medium under these conditions even though they are not growing. Thus, choZ cells have an Opi- phenotype only when deprived of ethanolamine or choline. This phenotype is relieved when choline or ethanolamine is added to the culture and PC biosynthesis is restored (Letts and Henry, 1985). The cho2 and opi3 mutants exhibit similar conditional Opi- phenotypes. Unlike choZ mutants, however, opi3 and opi2 mutants grow whether or not choline is present (Summers et al., 1988; McGraw and Henry, 1989). Despite the fact that the cells are growing, PC biosynthesis in ch02 and opi3 cells is considerably decreased when no supplement, such as choline, is present in the growth medium. With cho2 mutants, MME, DME or choline each enter the pathway for PC biosynthesis downstream of the genetic lesion and are thus capable of restoring PC biosynthesis (Summers et al., 1988;Fig. 2). In opij mutant strains, the biochemical defect resides in the final two methylation steps in PC biosynthesis and only choline is capable of restoring PC biosynthesis. The presence of DME in the growth medium restores synthesis of PDME, but does not restore synthesis of PC (Fig. 2). The precursor MME, however, enters the pathway upstream of the genetic lesion and is, therefore, incapable of restoring synthesis of PDME or PC (McGraw and Henry, 1989). Both opi3 and cho2 mutants are similar to choZ mutants in that they display an Opi- phenotype and an inability to repress IlPS expression in response to inositol when grown in the absence of a supplement capable of restoring PC biosynthesis. In both mutants, constitutive expression of IlPS has been shown to be due to constitutive expression of the ZNOZ transcript (Hirsch and Henry, 1986; McGraw and Henry, 1989). Thus, when cho2 cells are grown in the absence of MME, DME or choline, the ZNOZ transcript is expressed at the derepressed level whether inositol is present or not (Hirsch and Henry, 1986). In wild-type cells, in contrast, addition of inositol without choline or MME leads to a decreased expression of the ZNOZ transcript to 10% of the derepressed levels. The opi3 mutants are similar to ch02 mutants in the sense that the ZNOZ gene is not repressed in response to inositol alone. With the opi3 mutant, however, the presence of MME fails to restore PC biosynthesis and also fails to eliminate the Opi- phenotype or restore regulation of ZNOZ in response to inositol. Only addition of choline or DME eliminates the Opi- phenotype and restores IlPS regulation in an opi3 mutant (McGraw and Henry, 1989). Thus, with each of the mutants choZ, ch02 and opi3, only those growth conditions that restore synthesis of PC (or PDME synthesis) result in restoration of ZNOZ regulation in response to inositol. Since regulation of ZNOZ is restored in the opi3 mutant supplemented with DME (a growth condition that produces only PDME

32

MICHAEL J WHITE, JOHN M . LOPES AND SUSAN A HENRY

synthesis and not PC synthesis), synthesis of PDME is sufficient to restore regulation of I N 0 2 but synthesis of PMME is not (McGraw and Henry, 1989). Furthermore, regulation of INOZ can be restored whether PC is made directly via the Kennedy pathway (as in the opi3, cho2 or choZ mutants supplemented with choline), via methylation of PE (as in the choZ mutant supplemented with ethanolamine) or via methylation of PMME (as in the cho2 or cho2 mutants supplemented with MME). These results suggest that regulation of the INOZ gene in response to exogenous inositol is dependent upon a signal generated in the membrane only when PC (or PDME) is actively synthesized. Furthermore, it is clear that the regulatory response to PC biosynthesis is transmitted to the transcriptional apparatus that controls IN02 since the level of IN02 transcript is controlled by this mechanism (Hirsch and Henry, 1986; McGraw and Henry, 1989). VIII. The Regulatory Cascade Controlling IlPS and Other Coregulated Enzymes of Phospholipid Synthesis

That the enzymes listed in Table 1 show a common form of regulation in response to soluble precursors of phospholipid synthesis does not in itself demonstrate that they are under common genetic control. Evidence that a single regulatory cascade controls the entire set of coregulated enzymes has come from molecular, genetic and biochemical studies of strains bearing mutations defining regulatory genes. Originally, these regulatory mutations were identified on the basis of their effect upon regulation of IlPS. Later, it was shown that all of these regulatory mutations are pleiotropic, and exhibit altered regulation of the combined set of coregulated enzymes listed in Table 1. Two classes of regulatory genes have been identified, namely those that encode positive regulators, and those that encode negative regulators. All of the mutations characterized to date are genetically recessive and are, therefore, believed to have destroyed the functions of their respective gene products. Thus, a loss of function of a positive regulator is expected to result in the inability of a strain carrying such a mutation to express (or derepress) the gene products under control of the mutant regulatory gene. By similar reasoning, loss of function of a negative regulatory product would result in the inability of a strain bearing that mutation to repress the gene products under control of the mutant regulatory gene. Hence, a loss-of-function mutation in a gene encoding a negative regulatory factor would result in constitutive expression of the gene products under control of the regulatory gene in question. Two phenotypes corresponding to mutations in positive and negative

33

INOSITOL METABOLISM IN YEASTS

regulatory genes of phospholipid biosynthesis have been identified. Mutations defining the positive regulatory genes are unable to derepress IlPS and are, therefore, inositol auxotrophs (Ino-). Mutations defining the negative regulatory genes express IlPS constitutively at a high level and possess a phenotype of overproduction of inositol (Opi-). Such strains excrete inositol into the growth medium. The Opi- mutants were originally isolated in a screening procedure using a bioassay to detect excreted inositol (Greenberg et al., 1982b). However, all such mutant strains (both Ino- and Opi-) analysed to date are defective not only in regulation of IlPS but also in regulation of the other coregulated enzymes listed in Table 1 (Carman and Henry, 1989). This observation suggests that a single set of regulatory factors mediates the response to different concentrations of phospholipid precursors including inositol. A . POSITIVE REGULATORS,

IN02

AND

IN04

The I N 0 2 and I N 0 4 genes are genetically unlinked to the INOl gene and have been identified as regulatory genes whose wild-type products are necessary for expression (or derepression) of the I N O l gene product, IlPS (Donahue and Henry, 1981a; Loewy and Henry, 1984). Mutants bearing lesions at either of these two loci constitutively express repressed levels of IlPS and are, consequently, inositol auxotrophs (Ino-). When grown in medium containing low concentrations of inositol(l0 p ~ )a ,condition that allows partial derepression of IlPS in wild-type cells, in02 and in04 mutants fail to synthesize IlPS (Donahue and Henry, 1981a). Further characterization of several in02 and in04 mutant alleles has shown that these mutations are pleiotropic and express lower, but constitutive, levels of several phospholipid biosynthetic enzymes (Henry et al., 1984; Loewy and Henry, 1984; Bailis et al., 1987). Synthesis of PC via methylation of P E in the in02 and in04 mutant cells, for example, is considerably decreased (Loewy and Henry, 1984) and resembles synthesis of PC in wildtype cells grown under repressing conditions (i.e. in the presence of inositol and choline). Even under repressing conditions, wild-type strains synthesize more PC via the methylation pathway than do the in02 and in04 mutant strains (Loewy and Henry, 1984). All in02 and in04 mutant strains have phospholipids that contain significantly decreased proportions of PC. Approximately 40% of total phospholipid in wild-type cells is PC whereas, in in02 and in04 mutants, PC levels are decreased to 1&15% of the total phospholipids (Loewy and Henry, 1984). Phosphatidylserine synthase is also expressed at a lower constitutive level in in02 and in04 mutants. Levels of activity of this enzyme in in02 and in04 strains are intermediate between the fully repressed and fully derepressed wild-type levels regardless of

34

MICHAEL 1. WHITE, IOHN M. LOPES AND SUSAN A. HENRY

growth conditions (Bailis et al., 1987). In addition, CDP-DGS (Homann et al., 1987a) and PSD (E. Lamping and S. D. Kohlwein, personal communication) activity levels are not regulated in in02 or in04 mutant strains in response to phospholipid precursors. Hirsch and Henry (1986) reported that in02 and in04 mutations affect the steady-state levels of the IN02 message. Strains carrying in02 and in04 mutations express only repressed levels of IN02 mRNA, even when they are grown under conditions that allow derepression of the IN02 gene in wild-type cells (Hirsch and Henry, 1986). Bailisetal. (1987) have shown that in02 and in04 mutants also express repressed levels of CHOZ mRNA under both repressing and derepressing growth conditions. Thus, in02 and in04 mutants are defective in their ability to regulate many of the phospholipid biosynthetic enzymes that are subject to co-ordinate control by inositol and choline. For those enzymes whose structural genes have been cloned (IN02 and CHOI),it has been possible to show that the effect of the in02 and in04 mutations occurs at a transcriptional level. Thus, the I N 0 2 and I N 0 4 genes are believed to encode positive regulatory factors required for transcription of structural genes that are co-ordinately regulated in response to the presence of inositol and choline. Klig et al. (1988b) reported isolation of the positive regulatory gene I N 0 4 from a library of yeast genomic DNA that was screened by complementation of an in04 mutation. Two clones, each containing a 5.3 kbp overlapping region of homology, were found to restore the Ino+ phenotype to an in04 mutation. Immunoprecipitation studies, using IlPS-generated antibody, revealed that both of the two ino4-complementing clones are capable of restoring production and regulation of the 62 kDa IlPS subunit when transformed into in04 mutant strains. The plasmid carrying the I N 0 4 gene also restored synthesis of wild-type levels of methylated phospholipids to in04 mutants transformed with it (Klig et al., 1988b). Thus, the presence of the cloned IN04 DNA simultaneously restores to in04 mutants normal production and regulation of the IN02 gene product, as well as normal synthesis of PC. Genetic analysis, as well as Southern-blot analysis, of in04 mutants carrying integrated copies of the cloned DNA confirmed that it was derived from the genomic I N 0 4 locus. Hoshizaki et al. (1990) further subcloned the ino4-complementing DNA and sequenced a 1350bp fragment that retained the ability to complement in04 mutations. The IN04 gene is located within a fragment corresponding to a 453 bp open-reading frame and encoding a 600-nucleotide mRNA. A gene disruption was constructed, removing a 277 bp fragment from the 5' end of the open-reading frame and replacing it with a URA3 selectable marker. Using the one-step genedisruption procedure of Rothstein (1983), the disruption was integrated into the yeast genome and substituted for the wild-type locus. The phenotype of

INOSITOL METABOLISM IN YEASTS

35

the in04 deletion mutant was identical to other in04 mutants. It exhibited repressed levels of INOZ mRNA, decreased PC biosynthesis and had an Ino- phenotype. The ZN04 DNA sequence encodes a predicted protein (Ino4p) composed of 151 amino-acid residues with a molecular weight of 17,378. Ino4p is highly basic, largely hydrophilic and shows no evidence of having membranespanning regions (Hoshizaki et al., 1990). However, contained within Ino4p are regions of elevated hydrophobicity representing a-helical or P-sheet configurations. Potential protein phosphorylation sites at turns between ahelical or P-sheet regions of the Ino4p amino-acid sequnce are predicted by computer-aided analysis (Hoshizaki et al., 1990). The Ino4p amino-acid sequence was examined for structures associated with DNA-binding proteins. No sequence homologous to the helix-turn-helix motif (Brennan and Matthews, 1989; Mitchell and Tjian, 1989), the zinc-finger motif (Lee etal., 1989; Mitchell and Tjian, 1989; Rajavashisth et al., 1989) or the leucinezipper motif (Brendel and Karlin, 1989; Kouzarides and Ziff, 1989; Mitchell and Tjian, 1989) was detected. However, several regions containing homology to the Myc family of oncogene proteins and the lupus LA antigen protein were detected in the Ino4p sequence. In a 132 amino-acid residue overlap, Ino4p shows 25% identity to the lupus LA protein that is known to bind to several RNA molecules (Chambers et al., 1988). Moreover, a 74 amino-acid residue stretch of Ino4p shows approximately 30% identity with human and mouse N-myc proteins and the mouse L-myc protein. Contained within this region of homology are similarities to the amphipathic helixloop-helix motif that has been reported in Myc proteins (Murre et al., 1989). This motif has been postulated to play a role in protein dimerization and DNA binding. The similarities found between Ino4p and these two families of proteins may reflect a role for the I N 0 4 gene product in binding nucleic acids. The I N 0 2 gene has been cloned by screening a library of yeast genomic DNA for sequences capable of complementing in02 mutations (D. M. Nikoloff, personal communication). A gene-disruption mutation has been constructed using the cloned DNA; it has an Ino- phenotype and is biochemically indistinguishable from other in02 mutants. The DNA sequence of the ino2-complementing clone has been determined and found to encode a somewhat acidic (PI = 5.7) protein (Ino2p) composed of 304 amino-acid residues. A region of homology to the helix-loop-helix motif of the Myc family of proteins has also been detected in Ino2p (D. M. Nikoloff, personal communication). It is tempting to speculate that the helix-loophelix motif observed in Ino4p and Ino2p is involved in dimerization and interaction of the two proteins to form a protein complex that activates the ZNOZ promoter. However, much work remains to be done before definitive evidence supporting such a model can be produced.

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MICHAEL 1. WHITE, JOHN M. LOPES AND SUSAN A HENRY

B. NEGATIVE REGULATOR,

OPIl

The opil mutants represent one genetic complementation group from a very large collection of mutants possessing the Opi- phenotype (Greenberg et al., 1982b). As previously stated, Opi- mutants were originally detected on the basis of their inositol-excretion phenotype using a bioassay (Greenberg et al., 1982a). The opil mutants constitutively overexpress IlPS and a large number of other phospholipid-synthesizing enzymes (Homann et al., 1985, 1987a; Klig et a f . , 1985, 1988a; Bailis et al., 1987). These mutants are recessive and are not linked to the ZNOZ structural gene (Greenberg et al., 1982a). Based on genetic analysis, the OPIl locus is believed to encode a negative regulator of phospholipid biosynthesis. In opil mutants, IlPS is expressed constitutively at a level approximately two-fold higher than the wild-type derepressed level mutant. A similar Opimutant has been described in N. crassa (Schablik et a f . ,1988). This mutant, also termed opil , was isolated from slow-growing (inof+’-)spontaneous mutants of an inositol-requiring auxotroph (inof-). The opil mutation, when acting upon the wild-type inof+allele in N . crassa, caused an increase in IlPS expression. As we have previously stated, however, lesions in structural genes that encode enzymes involved in the PC biosynthetic pathway also cause expression of Opi- phenotypes in Sacch. cerevisiae (Greenberg et al., 1983; Klig et af., 1988a; Summers et a f . , 1988; McGraw and Henry, 1989). Mutants of Sacch. cerevisiae defective in PC biosynthesis, however, have a conditional Opi- phenotype that is displayed only under growth conditions that block PC biosynthesis. The Opi- phenotype in opil mutants is not influenced by the presence of choline in growth media. Since the opil mutant of N. crassa has not been tested for conditionality of its Opiphenotype, it is unclear as yet whether the opil mutation is analogous to the opil mutations found in Sacch. cerevisiae or whether it represents a lesion in one of the structural genes involved in PC biosynthesis. Strains of Sacch. cerevisiae carrying the opil mutation, in contrast to wildtype strains, have an unchanged phospholipid composition regardless of the presence or absence of phospholipid precursors. Furthermore, the relative rates of synthesis of various phospholipids are virtually unaffected by the presence of phospholipid precursors (Klig et af., 1985,1988a). In particular, addition of inositol to the growth medium does not lead to an increase in PI biosynthesis in opil cells, or to a coupled decrease in PS synthesis, as it does in wild-type cells (Klig et al., 1985; Kelley et al., 1988). In wild-type cells, PSS (Homann et al., 1987a; Klig e t a f . ,l985,1988a), CDP-DGS (Homann et a f . ,1985,1987a; Klig et a f . ,1988a) and the PMTs (Klig et a f . ,1985) are all repressed by the presence of phospholipid precursors in the growth medium. In opil mutants, in contrast, these same activities are expressed constitutively. Thus, the OPZZ gene product is believed to participate in co-ordinate

INOSITOL METABOLISM IN YEASTS

37

regulation of phospholipid biosynthesis. The hypothesis that the OPZZ locus encodes a negative regulator is further supported by the fact that IlPS, PSS and CDP-DGS actitivies are all constitutively overproduced in opil mutants. The OPZl gene product is believed to exert its influence at the transcriptional level since the ZNOZ transcript is constitutively overproduced in cells containing an opil mutation. When grown under repressing as well as derepressing growth conditions, opil mutants express two or three times more I N 0 1 mRNA than fully derepressed wild-type cells (Hirsch and Henry, 1986; White et al., 1991). In an opil background, the CHOZ transcript is also constitutively derepressed and is expressed at an elevated level (Bailis et al. , 1987). The OPZl gene in Sacch. cerevisiae has been mapped on chromosome VIII and lies adjacent to the SPOlZ gene (White et al., 1991). A 9.5 kbp yeast genomic clone containing the SPOZl gene (obtained from R. Esposito) was found to contain sequences capable of complementing the opil mutation. Further subcloning and complementation testing of fragments of the SPOll-containing DNA confirmed that the OPZZ gene is contained with a 2 kbp DNA fragment that is distinct from the SPOZZ gene (White et al., 1991). Thus, the OPZZ locus lies adjacent to the SPOZZ locus on chromosome VIII and was fortuitously cloned with the SPOZZ gene. The opil-complementing subclone was shown definitively to contain the OPZZ gene by creating disruption alleles with either an insertion (whereby a LEU2 selectable marker is inserted into the OPZZ coding region) or a deletion (whereby the whole coding region was removed and replaced with a LEU2 gene) mutation. These constructs were reinserted into the genome replacing the genomic wild-type sequence. Genetic analysis confirmed that the genedisruption events are linked to the OPZZ locus (White et al., 1991). The disruption alleles display an Opi- phenotype and express high constitutive levels of I N 0 1 mRNA under all growth conditions. With these characteristics, the disruption alleles of opil are indistinguishable from previously isolated opil mutants that had been isolated following chemical mutagenesis (Greenberg et al., 1982b). The protein (Opilp) predicted by translation of the OPZZ DNA sequence is composed of 404 amino-acid residues, corresponding to a molecular weight of 40,036. Based on its amino-acid composition, Opilp would appear to be a fairly acidic protein with a plvalue of 4.77. When Opilp was analysed for possible DNA-binding motifs, a heptad repeat of leucine residues, better known as a leucine zipper (Landschulz et al., 1988), was identified. NTerminal to this repeat in Opilp is a basic region consisting of 30 amino-acid residues. In conjunction with one another, these two motifs have been implicated in protein-protein interactions between DNA-binding proteins that regulate transcription (Brendel and Karlin, 1989; Kouzarides and Ziff,

38

MICHAEL J . WHITE, JOHN

M. LOPES AND SUSAN A . HENRY

1989). Whether Opilp dimerizes with itself to form a protein complex that binds to DNA as does the product of the yeast gene G C N 4 (Kouzarides and Ziff, 1989; O’Shea et al., 1989), or whether it complexes with another protein, as do the d u n and cFos proto-oncogene products (Kouzarides and Ziff, 1989), is unknown at present. Polyglutamine-residue stretches have also been detected in Opilp (White et al., 1991). While the significance of stretches of polyglutamine residues within a protein is unknown at present, it is noteworthy that similar aminoacid sequences have been reported in several other yeast proteins having regulatory functions (Schultz and Carlson, 1987; Passmore et al., 1988; Garrett and Broach, 1989). Gap-repair studies, DNA sequencing of existing opil alleles, and site-directed mutagenesis of the OPIl coding region will be employed to determine the function(s) of these features of Opi lp. C. EPISTATIC INTERACTION OF REGULATORY MUTATIONS

In order to understand the interaction of regulatory factors controlling phospholipid biosynthesis in yeast, the epistatic relationships of the ino2, in04 and opil mutations have been investigated. The basic premise of such an analysis is that the phenotype of the double-mutant strains may produce insights into interactions among the gene products. In some mutants, the phenotype produced by one of the two mutations may predominate over the phenotype produced by the other mutation in a double-mutant strain. In such a mutant, the mutation associated with the phenotype observed in the double mutant is said to be epistatic to the mutation whose associated phenotype is masked. For example, the haploid double-mutant strains with genotypes in02 opil , or in04 opil are all Ino- in phenotype (Henry et al., 1984; Loewy et al., 1986). Both double-mutant strains (in02 opil and in04 o p i l ) express repressed levels of IlPS. With or without the OPIl gene product, there is no derepression of IlPS in an in02 or in04 background. Thus, in04 and in02 mutations are said to be epistatic to opil mutations. This result implies that, in the absence of a functional OPIl gene product, the I N 0 2 and I N 0 4 gene products are still required for derepression of I N O l . By contrast, the OPIl gene product has no influence upon I N O l expression if the I N 0 2 and I N 0 4 gene products are mutated. These results can be used to evaluate several models for regulation of I N O l , CHOl and the other genes presumably under control of the regulatory cascade in which the I N 0 2 , I N 0 4 and OPIl gene products participate. The results of the epistasis studies are not compatible with any model in which the OPIl gene product functions more directly as a regulator of the I N O l gene than do the I N 0 2 and I N 0 4 gene products. The epistatic interactions are consistent, however, with a model in which the OPII gene

INOSITOL METABOLISM IN YEASTS

39

product functions as a negative regulator of the IN02 and IN04 genes which, in turn, serve as positive regulators of INOZ. In this model, ZNOl, and presumably CHOI and the other coregulated genes, would be predicted to be under positive regulation (Henry etal., 1984). As a test of this model, an attempt was made to isolate mutants defining negative regulatory genes whose products have a more direct effect on IN02 than products of the IN02 and IN04loci (Loewy, 1985). No such mutants were detected after an exhaustive search. As we discuss in the final sections of this review, however, there is evidence that IN02, IN04 and OPIl gene products may all interact directly with the INOI promoter. Furthermore, despite the genetic evidence already discussed, there is evidence suggesting that the INOl gene may be under negative control. D . EFFECTSOFTHEINO,?, I N 0 4 A N D OPIl GENE PRODUCTS ON TRANSCRIPTION OF IN02 AND OTHER GENES ENCODING PHOSPHOLIPID BIOSYNTHETIC FUNCTIONS

In the preceding discussion, we assumed that products of the IN04, IN02 and OPII genes are regulatory factors that have the ability to influence expression of a large set of unlinked structural genes. Obviously, then, it is important to characterize the mechanisms by which these regulatory factors interact with promoters of the structural genes under their control. The constitutively decreased levels of INOl and CHOI mRNA in in02 and in04 mutant cells imply that the IN02 and IN04 genes encode positive regulatory factors required for maximal transcription of IN02 , CHOI and other genes encoding phospholipid biosynthetic enzymes. The high constitutive levels of IN02 and CHOI mRNAs in opi2 mutant cells suggest that the OPI2 gene product is a negative regulatory factor required for repression of transcription of phospholipid biosynthetic genes. The results discussed thus far, however, do not provide evidence that these gene products interact directly with the promoters in question. In an analysis of the INOl 5' promoter region, Hirsch (1987) and J. P.Hirsch and J. M. Lopes (unpublished data) identified at least two sites in the IN02 promoter that act to decrease transcription under growth conditions that cause repression. In the study by Hirsch (1987), a portion of the 5' end of the I N 0 2 gene containing 543 nucleotide residues upstream of the start of transcription was fused, along with an initial portion of the INOI coding sequence, in frame to the lucZ reporter gene from E. coli.This fusion (to be referred to as the -543 fusion) is fully regulated when reintroduced into the yeast genome by integrative transformation. That is to say, expression of P-galactosidase from the -543 fusion is repressed about 10-fold in response to the presence of inositol as the sole supplement in the growth medium. Expression of the

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MICHAEL J. WHITE, JOHN M LOPES AND SUSAN A. HENRY

-543 fusion is repressed another two- to three-fold (for a total of 20- to 30fold) in response to the presence of inositol together with choline. Additional constructions were made, starting with the fully regulated construct and deleting sequentially larger portions of the 5’ end of the I N 0 2 promoter. Each of these constructs will be referred to by the nucleotide at which the deletion ends (i.e. the construct containing 543 nucleotides will be called fusion -543, meaning that the end-point of the I N 0 2 promoter sequence is 543 nucleotides 5’from the start of transcription). All constructs were reintegrated into the genome in single copy. The level of ZNOl ‘lacZ fusion mRNA produced by these constructs, under repressing and derepressing growth conditions, was analysed by several methods in order to define sequences required for INOl transcription and regulation. A fusion containing 333 nucleotides (-333 fusion) of sequence 5’ from the start of transcription of the I N 0 2 gene is also fully regulated. However, deletion of a site located between nucleotides -333 and -259 from the start of transcription (to produce the -259 fusion) leads to elevated expression of the I N 0 1 ‘ZacZ reporter gene under repressing conditions. The -259 fusion is still repressed about four-fold in the presence of inositol. Deletion of a site located between -259 and -213 leads to two- to 2.5-fold elevated expression under the derepressed conditions. Deletion of sequences between - 153and - 120 leads to progressively higher levels of expression of the construct under repressing growth conditions. The -120 fusion is, thus, expressed constitutively at the derepressed level. The fact that removal of sequences 5’ to nucleotide - 120leads to constitutive expression implies that the I N 0 2 promoter is under negative control. Another control element that is required for I N 0 2 expression was discovered between nucleotides - 120 and -86 and is believed to be a TATA promoter element (Nagawa and Fink, 1985). Expression of three INOl promoter-deletion/facZ fusion constructions was further analysed in several genetic backgrounds. These constructions include the fully regulated fusion (-543) as well as two deletions containing 213 and 120 nucleotide residues (-213 and - 120 fusions) of sequence 5‘ to the start of transcription. Expression of these two deletion constructs and the fully regulated -543 fusion was studied following integration in single copy into wild-type, o p i l , in02 and in02 in04 genetic backgrounds (J. P. Hirsch and J. M. Lopes, unpublished data). The -543 fusion construct, as stated previously, is fully regulated in response to inositol and choline when transformed into wild-type strains. Expression of the -213 fusion is only partially repressed in the presence of inositol and choline, and is expressed at a two- to 2.5-fold higher level under derepressed conditions in the wildtype background. The - 120 fusion is constitutively derepressed under all growth conditions in a wild-type background. In an opil background, all

INOSITOL METABOLISM IN YEASTS

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three fusions (-543, -213 and -120) are expressed constitutively. In an in02 or in02 in04 strain, the fully regulated -542 fusion is expressed at extremely low levels. In this regard, it is identical to the native I N 0 1 mRNA, which is expressed at repressed levels in an in02 or in04 genetic background. The -213 and -120 fusions, however, are expressed at derepressed levels in the presence or absence of phospholipid precursors in the in02 and in04 genetic backgrounds. Their expression is, therefore, not dependent on activation by the I N 0 2 and I N 0 4 gene products. A computer-assisted examination of the INOZ promoter (Carman and Henry, 1989; J. P. Hirsch and J. M. Lopes, unpublished data) and the 5’ region of-three other structural phospholipid genes (CHOZ, C H 0 2 and OP13; Carman and Henry, 1989) has revealed a repeated nine-nucleotide element with a consensus 5’-ATGT(G/T)AA(A/T)T-3’.In the INOZ 5‘ promoter region, this sequence was found to be repeated seven times with one repeat being present downstream of the TATA element. The 5‘ deletion analysis performed by J. M. Lopes (unpublished data) resulted in a progressive removal of these nine-nucleotide elements with a concurrent decrease in regulation by inositol. The -120 fusion construct containing a deletion of all sequences 5’ to nucleotide -120 expresses derepressed levels of INOl’facZ mRNA constitutively, and is missing all of the repeated elements upstream of the TATA promoter element. Whether these ninenucleotide repeats define sites for binding of a negative regulatory factor(s), or whether they represent positive promoter elements which are not responsible for gene regulation, is at present unknown. However, the fact that these conserved sequences are present in the upstream region of other phospholipid structural genes (Carman and Henry, 1989) that are coordinately regulated in response to inositol provides support for the idea that these elements may be involved in regulation or expression of these genes. Based on the identification of cis-acting regulatory elements in the 5’ untranslated region of INOZ J. M. Lopes (unpublished data) constructed a series of overlapping DNA templates from the 5‘ promoter region of I N 0 1 . These templates were employed in DNA-binding/mobilityshift assays and oligonucleotide-competition experiments to assess possible interaction of DNA-binding proteins. When a template from the INOZ promoter region comprised of nucleotides -333 to -259 was used in binding experiments, two DNA-protein complexes were formed that are competed for by a 21 bp oligonucleotide that contains within it the conserved ninenucleotide sequence 5’-ATGTGAAAT-3’. The DNA-binding activity that recognizes the nine-nucleotide repeat is present in wild-type extracts and in extracts prepared from opiZ, in02 and in04 mutants. Another complex formed on the -333 to -259 template was absent from extracts derived from strains carrying the opiZ mutation, but was present in in02, in04 and

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wild-type extracts. A second template, spanning nucleotides -259 to - 155, forms at least three DNA-protein complexes with proteins present in extracts of wild-type cells. One complex formed on the template is competed for by the oligonucleotide containing the 9 bp repeat. The other two complexes are present in extracts prepared from opiI and wild-type strains, but are absent from in02 and in04 mutant extracts. The ZNO2-, ZN04dependent DNA-protein complexes are not subject to competition by the oligonucleotide containing the 9 bp repeat. The analysis by J. M. Lopes (unpublished data) has detected at least three DNA-binding activities capable of recognizing sequences in the I N O l promoter region. A protein or proteins capable of forming a complex that is competed away by an oligonucleotide containing the 9 bp repeat recognizes sites distributed on both templates. A protein or proteins capable of forming a complex with the template containing the I N O l promoter sequence from nucleotides -333 to -259 is present in wild-type and in02 and in04 extracts, but is missing from extracts prepared from the opil mutant. A protein (or proteins) absent from in02 and in04 extracts forms two complexes on the template spanning nucleotides -259 to -155. As to whether the existing regulatory genes encode factors that bind directly to the ZNOl promoter, and possibly other structural genes involved in phospholipid biosynthesis, to control transcription, or whether they are involved in regulation of other effectors, remains to be seen. Identification and isolation of OPZl, I N 0 2 and I N 0 4 gene products combined with further DNA-binding studies will be necessary to resolve these questions. Recently, it has also been shown that transcription of the I N 0 1 gene is highly sensitive to perturbations in the general transcription apparatus. Arndt el al. (1989) isolated a series of yeast mutants defining lesions in the RNA transcription apparatus and found that some of these mutants had an Ino- phenotype due to an inability to derepress the ZNOl gene. Furthermore, Nonet and Young (1989) found a similar phenotype in a different set of mutants possessing partial deletions of the carboxyl-terminal repeat of the large RNA polymerase subunit. These mutants are Ino-, and also have temperature- and cold-sensitive phenotypes. Similar pleiotropic phenotypes were detected in the mutants described by Arndt et al. (1989). In addition, Nonet and Young (1989) isolated second-site suppressor mutations capable of suppressing the phenotypes of their RNA polymerase mutants. The second-site suppressor mutation SRB2-I was isolated, mapped, and a partial deletion allele, srbA10, was constructed. The strain bearing the srb A 10 deletion also proved to be temperature sensitive, cold sensitive and Ino-. It is believed that the SRB2 locus also encodes a factor involved in RNA transcription that interacts with the large subunit of RNA polymerase. The Ino- phenotype of these mutants is believed to be due to the particular

.

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nature of the interaction of ZNOZ-specific regulatory factors with the RNA transcription apparatus (Scafe et al., 1990). It is important to note that the ZN02, I N 0 4 and OPZI mutations clearly define specific regulators and not general transcription factors. Deletion mutants for each of these genes have phenotypes similar to the originally isolated point mutants, and exhibit defects only in phospholipid biosynthesis. The ino2, in04 and opil deletion mutants are not cold- or temperature-sensitive, nor do they appear to have any other growth defect. E. A MODEL FOR REGULATION OF PHOSPHOLIPID SYNTHESIS BY INOSITOL AND OTHER PHOSPHOLIPID PRECURSORS

In the preceding sections of this review, we presented evidence showing that phospholipid synthesis in yeast is regulated in a co-ordinated fashion. A number of enzymes (Table 1)are subject to this regulation. The key features of the regulation are as follows: (1) Each of the coregulated enzyme activities is partially repressed by addition of inositol to the growth medium. Addition of choline together with inositol produces further repression. For some enzymes, other precursors such as ethanolamine or serine will substitute for choline in producing further repression of enzyme activity in the presence of inositol. (2) Mutations in the regulatory genes ZN02, I N 0 4 and OPZZ have pleiotropic effects, influencing expression of most, if not all, of the coregulated enzyme activities. The regulatory genes are not genetically linked to each other and they are not linked to structural genes encoding the enzymes. Furthermore, the structural genes that have been studied at a genetic level are not linked to each other; they are scattered throughout the genome. (3) For those structural genes that have been cloned, regulation in response to inositol and choline occurs at the level of transcript abundance. Furthermore, mutations in the I N 0 2 , I N 0 4 and OPZZ genes affect expression of the structural genes at the level of transcription. Cloning and DNA sequencing of the regulatory genes have produced additional evidence that these genes encode DNA-binding proteins. In addition, molecular studies of the promoter regions of several of the coregulated structural genes have provided evidence that sequences 5' to these genes mediate regulation in response to precursors and regulatory genes. (4) Initial studies of genetic epistasis suggested that the ultimate regulation of the ZNOZ gene would be positive. However, a detailed analysis of the ZNOZ promoter has produced evidence for both positive and negative regulation of ZNOZ transcription. ( 5 ) Studies of expression of the I N 0 1 gene suggests that transcriptional regulation in response to inositol is dependent upon ongoing synthesis of methylated phospholipids, specifically PC or PDME. (6) Biochemical studies on PSS activity in vitro suggest that this

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MICHAEL J . WHITE, JOHN M. LOPES AND SUSAN A. HENRY

C

U

FIG. 4. Proposed model for regulation of phospholipid synthesis by inositol in Saccharomyces cerevisiae. Phospholipid abbreviations are listed at the beginning of this chapter. Gene designations are explained in the text. Inositol which enters the pathway for phospholipid synthesis is produced from IlP which is synthesized endogenously from G6P or phospholipid turnover. The presence of exogenous inositol leads to elevated rates of PI biosynthesis (indicated by (+) in this reaction). It also leads to an immediate decrease in PS biosynthesis (indicated by (-) in this reaction). Other reactions in the cell are subject to repression (-) or induction (+) under these conditions. Thus, the presence of inositol leads to immediate and longterm changes in the pattern of phospholipid synthesis. The presence of inositol only leads to repression of the IN01 gene and its product, IlPS, when PC (or PDME)

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enzyme is inhibited non-competitively by free inositol. Furthermore, in vivo studies on relative rates of phospholipid biosynthesis suggest that the presence of inositol in the growth medium leads to a rapid shift in the flux of CDP-DG precursor in the direction of PI biosynthesis at the expense of PS biosynthesis. This reduction in PS biosynthesis necessarily leads to a reduction in available substrate for the reaction sequence leading from PS to PE, and, ultimately, to PC. The presence of inositol ultimately leads to repression of PSS activity at the level of expression of its subunit, repression of CDP-DGS activity, and induction of PAP. These considerations lead us to propose the following concerted model for regulation of phospholipid synthesis in response to inositol and other precursors (Fig. 4). According to this model, the signal transmitted to the transcriptional apparatus is not generated directly by the free precursors. Rather, the signal is generated by participation of these precursors in ongoing phospholipid synthesis. The presence of inositol, in particular, appears to be detected initially by the cell when increasing concentrations of inositol lead to increased PI biosynthesis at the expense of PS and, ultimately, PC biosynthesis. Evidence for this interpretation comes from biochemical studies on PS biosynthesis as well as analysis of IlPS regulation in mutants defective in PC biosynthesis. When PC (or PDME) biosynthesis is interrupted, the cell is incapable of responding to the presence of inositol to regulate the IN01 gene. However, the presence of inositol can be detected even when the continuity of the reaction sequence from PS through PE to PC is interrupted by a mutation, but only if PC biosynthesis is occurring by some route. Studies of I N 0 1 regulation in opi3 and cho2 mutants reveal that regulation of INOI is restored whether PC biosynthesis FIG. 4 (continued) biosynthesis is occurring. Hence, a signal of unknown mechanism must be generated within the membrane or at its surface. The signal must then be transmitted via an unknown number of steps to the transcriptional apparatus within the nucleus. Regulatory factors, presumably DNA-binding proteins, known to function within this network are OPII-, IN02- and IN04-dependent gene products. Another factor (designated R in this diagram) is known to interact with a 9 bp sequence that has been found repeated several times in the INOI promoter (J. P. Hirsch and J. M. Lopes, unpublished data). This same 9 bp repeat is also found in promoters of other coregulated genes (Carman and Henry, 1989). In this representation, R is portrayed as a repressor, possibly interacting with the OPII gene product to facilitate repression. The I N 0 2 and I N 0 4 gene products are shown as elements of a single complex whose role is to cause positive expression of the structural genes. In this model, the OPIl and R proteins are shown as interfering with the binding of the IN02/IN04 complex, thus inhibiting expression. Of course, this representation is strictly speculative. The true roles of these regulatory factors with regard to interactions among themselves and their involvement with sequences within the promoters of structural genes encoding phospholipid biosynthetic enzymes remain to be established.

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occurs via the de novo methylation of PE or via the CDP-choline (Kennedy) pathway. Thus, participation of inositol in phospholipid biosynthesis must be capable of producing a signal via either route. The fact that inositol causes induction of PAP activity, thus, potentially enhancing substrate availability on the Kennedy pathway, is consistent with this hypothesis. However, much less is known about regulation of enzymic activities on the Kennedy pathway than is known about regulation of the PE methylation pathway. The signal generated by the presence of inositol via synthesis of PC (or PDME) is transmitted to a regulatory network capable of regulating transcriptional activities of numerous unlinked structural genes. Three of the regulatory genes encoding proteins participating in this cascade have been identified and characterized on a molecular level. They are the " 0 2 , IN04 and OPIZ genes. Preliminary evidence suggests that these genes may encode DNA-binding proteins capable of interacting with specific sites in promoters of the coregulated genes. DNA-protein complexes dependent on the presence of the wild-type copies of each of the three genes have been detected using DNA templates derived from the I N 0 1 promoter. The OPIZ-dependent protein(s) binds to a region 5' to the region where the IN02-, ZNOCdependent complex forms on the ZNOZ promoter. Deletion analysis of the IN01 promoter fusions to lucZ showed that removal of sequences in the vicinity of the OPZI site leads to elevated expression under repressing conditions. Deletion of sequences in regions of the ZN02-, I N 0 4 dependent site leads to constitutive expression of the IN01 gene even in the absence of IN02 and I N 0 4 gene products. Furthermore, another factor that recognizes a 9 bp repeat in the IN02 promoter has been detected. This protein(s) is present in extracts of wild-type, ino2, in04 and opil cells. A 9 bp repeat occurs in multiple copies in the ZNOZ promoter as well as in the promoter of other coregulated genes. Removal of successive copies of this repeat from the ZNOZ promoter leads to a loss of repression, suggesting that the 9 bp repeat may be the binding site for a repressor. These data collectively suggest that multiple transcription factors are involved in regulation of the I N 0 1 gene. Both positive and negative sites appear to be located in the 5' region of the IN01 gene and, presumably, in promoters of other coregulated genes. The pattern of regulation described here is highly complex and will require much further analysis before it is fully understood. The model (Fig. 4)is, of course, highly speculative and is offered simply as a basis for further discussion and analysis.

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IX. Summary

Because of its accessibility to genetic and molecular studies, Succh. cerevisiue is an attractive organism in which to pursue studies of the complex roles of phosphoinositides and other inositol-containing metabolites. Biochemical studies have clearly demonstrated that PI, PIP, PIPz and the inositol phosphates derived from them exist in Succh. cerevisiue. It is clear that they are synthesized and turned over following pathways similar to those described in higher eukaryotes. Recent studies on yeast have also suggested that inositol phospholipids may play roles in complex signalling pathways similar to those detected in animal cells. In addition, inositol has been demonstrated to function in yeast as a global regulator of phospholipid synthesis. This regulation occurs on a transcriptional level and is highly complex. It is not yet known whether similar inositol-mediated regulation of phospholipid synthesis occurs in other eukaryotes.

X. Acknowledgements The authors wish to thank Susan L. Haslett for expert secretarial assistance in preparing the manuscript. This work was supported by grant GM 19629 from the National Institutes of Health to SAH. JML is supported by NIH Postdoctoral Fellowship GM 12099. REFERENCES

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