Biosynthesis of Inositol in Yeast · variety of neurotransmitters, hormones, and growth factors (for review, see Ref. 1). In the yeast Saccharomyces cereuisiae, synthesis of the phospholipid

THE JOURNAL OF BIOLOGICAL CHEMISTRY Q 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 264, No. 2, Issue of January 15, pp. 1274-1283,1989 Printed in U. S A .

Biosynthesis of Inositol in Yeast PRIMARY STRUCTURE OF MYO-INOSITOL-1-PHOSPHATE SYNTHASE (EC 5.5.1.4) AND FUNCTIONAL ANALYSIS OF ITS STRUCTURAL GENE, THE INOl LOCUS*

(Received for publication, June 6, 1988)

Margaret Dean-Johnson$ and Susan A. Henrye From the Departments of Genetics and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461

A biochemical, molecular, and genetic analysis of the Saccharomyces cerevisiae I N O l gene and its product, L-myo-inositol-1-phosphate synthase (EC 5.6.1.4) has been carried out. The sequence of the entire I N O l gene and surrounding regions has been determined. Com- puter analysis of the DNA sequence revealed four potential peptides. The largest open reading frame of 553 amino acids predicted a peptide with a molecular weight of 62,842. The amino acid composition and amino terminus of purified L-myo-inositol-l-phosphate synthase were chemically determined and compared to the amino acid composition and amino terminus of the protein predicted from the DNA sequence of the large open reading frame. This analysis established that the large open reading frame encodes L-myo-inositol-1-phosphate synthase. The largest of several small open reading frames adjacent to I N O l predicted a protein of 133 amino acids with a molecular weight of 15,182 and features which suggested that the encoded protein may be membrane-associated.

A gene disruption was constructed at I N O l by elim- inating a portion of the coding sequence and replacing it with another sequence. Strains carrying the gene disruption failed to express any protein cross-reactive to antibody directed against L-myo-inositol-l-phosphate synthase. Although auxotrophic for inositol, strains carrying the gene disruption were completely viable when supplemented with inositol. In a similar fashion, a gene disruption was constructed in the chromosomal locus of the 133-amino acid open reading frame. This mutation did not affect viability but did cause inositol to be excreted from the cell.

Recently, studies which encompass many aspects of animal, plant, and yeast cell physiology have shown that the biosynthesis and metabolism of inositol and the inositol phospholipids play central roles in signal transmission for a wide

* This work was supported in part by Grant GM-19629 (to S. A. H.) from the National Institutes of Health. This report was taken in part from a Ph.D. thesis submitted in 1987 by M. D. J. to the Albert Einstein College of Medicine. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) 5044.53.

$ Supported by National Institutes of Health Training Grant GM- 07128. Present address: Dept. of Biology, Yale University, New Ha- ven, CT 06520.

Present address: Dept. of Biological Sciences, Carnegie Mellon University, 4400 Fifth Ave., Pittsburgh, PA 15213. To whom reprint requests should be addressed.

variety of neurotransmitters, hormones, and growth factors (for review, see Ref. 1).

In the yeast Saccharomyces cereuisiae, synthesis of the phospholipid precursor inositol is one of the most highly regulated aspects of phospholipid metabolism. Regulation centers on the cytoplasmic enzyme MI-1-P synthase.’ MI-1- P synthase catalyzes the synthesis of inositol 1-phosphate de novo via the cyclization of glucose 6-phosphate (2-4). While the precise reaction mechanism has not been elucidated, the absolute requirement for NAD with no net gain in NADH (5) suggests that the overall reaction consists of a tightly coupled oxidation and reduction. Two intermediates, 5-ketoglucose 6- phosphate and inosose 2,1-phosphate, have been proposed (5- 7), but neither has been directly identified and each is assumed to be tightly bound to the enzyme (5, 8).

The molecular weight of the native enzyme in yeast as determined by gel filtration is approximately 240,000 (9). A single subunit of 62,000 is detected upon sodium dodecyl sulfate gel electrophoresis of the purified enzyme (9). Based upon an immunological analysis of inol mutants, the INOl locus was identified as the probable structural gene encoding the MI-1-P synthase subunit in yeast (9).

In yeast, MI-1-P synthase is regulated both in response to exogenous inositol and to unlinked regulatory genes (9-16). The addition of inositol to logarithmically growing cultures results in a 50-fold, time-dependent decrease in the enzyme’s activity. Immunological and biochemical evidence supports the conclusion that the pathway for inositol biosynthesis in yeast is regulated by the repression of enzyme synthesis (9). The S. cereuisiae INOl gene, which encodes MI-1-P synthase, was isolated by genetic complementation (12). The cloned gene was shown to complement two independently isolated allelic mutations at INOl (inol-5 and inol-13) (12).

The cloned INOl DNA was used as a probe to examine the expression of the gene under conditions known to regulate phospholipid synthesis (16). RNA blot hybridization detected two RNA species of 1.8 and 0.6 kb (16). Complementation analysis showed that the 1.8-kb RNA encodes the IN01 gene product (16). The level of RNA was repressed 30-fold when the cells were grown in the presence of inositol and choline together (16). The level of the 0.6-kb RNA is affected to a lesser degree by many of the same factors that influence INOl expression (16). A similar pattern of regulation has been shown for other enzymes of phospholipid biosynthesis in S. cereuisiae (17-22) (Fig. 1).

Mutations at loci unlinked to INOl are also known to affect expression of MI-1-P synthase. The opil mutant, identified on the basis of a bioassay for inositol excretion (141, causes the MI-1-P synthase subunit to be constitutively overex-

The abbreviations used are: MI-1-P synthase, L-myo-inositol-l- phosphate synthase; kb, kilobase(s).

1274

Primary Structure of Yeast myo-Inositol-1 -phosphate Synthase 1275

pressed. Other phospholipid biosynthetic enzymes which are coordinately regulated by inositol and choline are also constitutively expressed in the opil mutant strain (17,22). The in02 and in04 mutations, which lead to an inability to derepress MI-1-P synthase (9,16), likewise have pleiotropic effects upon the other enzymes which are regulated by inositol and choline (15, 22).

The analysis of the IN01 structural gene presented in this report provides the physical foundation needed to dissect the

PA > nr.

FIG. 1. Phospholipid biosynthesis in S. cerevisiae. Shown are reactions in the cytoplasm and the membrane which are involved in the synthesis of the major phospholipids and their precursors in S. cereuisiae. Steiner and Lester (23-25) detected most of these reactions in vitro in isolated S. cereuisiae membranes. Waechter and Lester (26,27) reported the synthesis of phosphatidylcholine (PC) via meth- ylation of phosphatidylethanolamine (PE) in S. cereuisiae membranes. Kennedy and Weiss (28) described the formation of phosphatidylethanolamine, phosphatidylcholine, phosphatidylmonomethyl- ethanolamine (PMME), and phosphatidyldimethylethanolamine (PDME) from exogenous precursors. The cytoplasmic synthesis of MI-1-P from glucose 6-phosphate in S. cereuisiae was described by Culbertson et al. (10). The assignment of genes known with certainty to be structural genes (ZNOI, CHOI) are shown by a gene designation above given reactions. Reactions subject to coordinate regulation by inositol and choline include: the formation of inositol 1-phosphate from glucose 6-phosphate, the formation of cytidine diphosphate diacylglycerol (CDP-DG) from phosphatidic acid (PA), the formation of phosphatidylserine (PS) from cytidine diphosphate diacylglycerol, the formation of phosphatidylethanolamine from phosphatidylserine, and the three sequential methylations of phosphatidylethanolamine to form phosphatidylcholine. The formation of phosphatidylinositol from cytidine diphosphate diacylglycerol is known to be constitutive (18). Z, inositol; G-6-P, glucose 6-phosphate; and I-I-P, inositol 1- phosphate.

complex regulatory network involved in the coordinate regulation of phospholipid biosynthesis in yeast (Fig. 1).

MATERIALS AND METHODS

Yeast and Bacterial Strains-Yeast strains which were used for the purification of MI-1-P synthase, preparation of yeast RNA, and gene disruptions are shown in Table I as are bacterial strains used for isolation of plasmid DNA.

Chemical Analysis of MI-1-P Synthase-Purification of MI-1-P synthase was carried out as described previously by Donahue and Henry (9). MI-1-P synthase purified by column chromatography was assayed by the rapid chemical method of Barnett et al. (29). The purified enzyme was tested for glycosylation' using the Schiff base reaction. The enzyme was not found to be glycosylated.

To determine the amino acid composition of purified MI-1-P synthase, the enzyme was dialyzed for 2 day 3 in the cold room against 5% acetic acid with three changes of this solution followed by dialysis against distilled water. Two hundred micrograms of protein were used for the analysis of the amino acid composition. The amino acid composition was determined using the methods of Steinman (30).

To sequence the amino terminus of MI-1-P synthase, purified enzyme was subjected to electrophoresis on 10% polyacrylamide gels under fully dissociating conditions (31,32), and the M, 62,000 subunit was reisolated from the gel (32). The amino terminus of the protein isolated from the gel was analyzed by automated Edman degradation. The work was contracted to the Protein Structure Laboratory at the University of California, Davis, CA. Two different reverse phase high performance liquid chromatography systems were used to identify the phenylthiohydantoin-amino acids. The first high performance liquid chromatography system employed the Waters Model 6000 gradient system with a WISP autosampler and a fixed wavelength detector. The second system was a Perkin-Elmer Series 4 gradient system with a Kratos 783 programmable variable. The sample supplied consisted of approximately 2.5 PM of the 62,000-dalton subunit of MI-1-P synthase. Approximately 50% of the sample was used in the analysis with 4% recovery estimated at the sequence level. Neither separation system provided sufficient resolution of serine and threonine deriva- tives to permit their unambiguous identification in the experimental sample.

Phospholipid Analysis-To determine the phospholipid composition of yeast strains carrying gene disruptions, phospholipids were labeled in uiuo to steady state with [32P]orthophosphate and extracted as described previously (15, 18). Phospholipids were separated chro- matographically in two dimensions on silica-impregnated paper using the methods of Steiner and Lester (24).

DNA Sequencing-The method of Henikoff (33) was used to gen-

TABLE I Genotypes and origins of yeast and bacterin strains which were employed in these studies

All yeast strains were routinely grown at 30 "C. Bacterial strains were grown at 37 "C. Strains Genotype Source or reference Use

Yeast d e 5 (Wild-type)

opil (Over-producer of inositol)

W303-1A

DC5 MC13

DC5D

Bacterial HblOl

JA300

MATa, d e 5

MATa, opil

MATa, leu2,-3112, trpl- 1, canl-100, u r d - I , ade 2-1, his3-11,15

Mat leu2-3,112, his 3 MATa, leu 2-3,112, inol-

13 MATaIMATa, leu2-

3.1 12/leu2-3, 112, his3/+, +lade2

F-, hsdS20 (TB-, mB-), recA13, ara-14, leuB6, proA2, lac YI , galK2, rpsL20 (Sm*), XY"~, mtl -I, supE44, X-

F-, thi-I, leuB6, supE44

Culbertson and Henry (10) Isolation of MI-1-P

Greenberg et al. (13, 14) Isolation of MI-1-P

Dr. Rodney Rothstein Isolation of yeast RNA

synthase

synthase

Dr. James Broach Gene disruption Klig and Henry (12) Complementation

Dr. Patricia McGraw Gene disruption analysis

Laboratory strain

Laboratory strain

Isolation of plasmid DNA

Isolation of plasmids used for gene disruptions

1276 Primary Structure of Yeast myo-Inositol-1 -phosphate Synthase erate and clone deletions well suited for the sequencing of long stretches of DNA. Primers synthesized for sequencing specific regions of the INOl gene were prepared by the oligonucleotide facility at the Albert Einstein College of Medicine. Sequencing packs supplied by New England Biolabs, Inc. were used for dideoxy sequencing (34,35) of the INOl gene and surrounding regions. Fig. 2 shows a partial restriction endonuclease map of the DNA fragment carrying the INOl gene, the smaller adjacent open reading frame, and surrounding regions. Arrows indicate the subclone, direction, and the strategy for sequencing. The length of each arrow represents the approximate extent of sequence information obtained.

The DNA Inspector I1 program (Textco, West Lebanon, NH) designed for the Apple Macintosh computer was used to analyze the DNA sequence for open reading frames, amino acid sequence, peptide molecular weight, codon usage, amino acid composition, hydrophilicity, and charge of the protein at pH 7.0. Bionet's computer program was used to search the National Biomedical Research Foundation Protein Identification Resourse database for proteins homologous to MI-1-P synthase and the small open reading frame upstream of the INOl gene. The DNA Inspector I1 program was used to compare DNA sequences for regions of similarity.

Construction and Analysis of Gene Disruptions-To determine whether or not the INOl gene or the adjacent small open reading frame is essential for the viability of the yeast cell, a one-step gene disruption was performed using the method described by Rothstein

disruptions. The method of Southern (38) was used to locate the (37). Fig. 3 outlines construction of the plasmids used for gene

LEU2 gene at the IN01 locus. Gene disruptions were carried out in a diploid strain DC5D (shown in Table I) which is homozygous for the leu 2-3, 112 mutant alleles but wild type at the INOl locus. Following transformation, this strain was sporulated and subjected to tetrad analysis to produce haploid strains carrying the gene disruption. Cellular extracts prepared from cultures grown from spore colonies from the tetrads were subjected to Western blot (39) analysis to assess the expression of MI-1-P synthase and Northern blot analysis (40) to assess the expression of the transcript from the small open reading frame.

RESULTS Sequence of the INOl Gene and Surrounding Regulatory

Regions-The DNA sequence of a 4176-base pair fragment,

>

301 307 CK " - Asp 16

c-

301 301 31918 ~ Asp 10 - - - del 68 "

sst-Bgl Asp 24 c"

SK 301 del 38 12- Asp 15 - ~ 301 2 307 - Asp 100

306 de144 del 1 PK

307 18

PB 20 "

RV 5

" PB 21 RV 29

I

PB 117 RV 21 "

PB 102

PB 106 - -1Kb -

FIG. 2. Sequencing strategy. Restriction fragments derived from plasmids YEpINOl and YIpINOl(12) were cloned into plasmids pUClS and pUC19 (36). The relevant restriction sites of the yeast sequences in YIpINOl DNA are shown at the top. The arrows and names above them indicate the subclone, the direction, and extent of sequence information obtained.

, 1

J

TAT TAG TATAAA TAA TGA

Smolt ORF

IN01 gene

420 bp 1650 bp

I 2.2 Kb

LEU 2gene

U - 1Kb-

FIG. 3. Construction of gene disruptions. A subclone which contains the unmethylated CM-Kpnl fragment of the INOl gene in plasmid pUC19 was used to disrupt the INOl gene. The 2.2-kb LEU2 gene was inserted into the plasmid following the removal of 663 base pairs from the coding region of the INOl gene using the restriction endonuclease EcoRV. Subsequently, the plasmid was cut with NcoZ and PvuI to release the fragment containing the LEU2 gene embedded in the remaining INOl DNA. The fragment was purified and used for transformation of a wild-type diploid. In a similar manner, 362 base pairs from the coding region of the small open reading frame adjacent to INOl were replaced with the 2.2-kb LEU2 gene. Frag- ments containing LEU2 which were derived from these constructions were transformed into diploid strain DC5D (Table I) in order to replace existing genomic sequences according to the method of Roth- stein (37). Gene replacement was confirmed by Southern blot analysis (38).

which is known from transformation, complementation, and molecular analyses (12, 16) to contain the entire INOl gene and surrounding regulatory regions, was obtained by analysis of both strands. Fig. 4 contains the sequence of the DNA fragment containing the INOl gene and surrounding region. Computer analysis revealed a large open reading frame which corresponds to the INOl gene (data to support this identification will be presented below). Three additional smaller open reading frames were detected in the adjacent DNA (Fig. 5).

The nucleotide sequence of the DNA fragment containing the INOl gene begins 1380 bases upstream of the largest open reading frame (Fig. 4). The peptide predicted from the sequence of the large open reading frame consists of 553 amino acids. There is a larger open reading frame starting at position 1315. The protein potentially encoded by this larger open reading frame has 22 additional amino acid residues. However, as will be discussed below, analysis of the amino terminus of inositol 1-phosphate synthase confirmed that position 1380 corresponds to the start of translation of inositol 1-phosphate synthase. Consequently, all subsequent analysis of the sequence was performed on the basis of the protein starting at position 1380. The codon usage in the INOl gene and its predicted amino acid composition are shown in Table 11. The computer analysis revealed that the codon usage was consistent with a moderately expressed yeast protein (41,42). At pH 7.0, the INOl protein has a charge of +2. A computer- generated hydrophilicity plot for the INOl gene product is shown in Fig. 6.

Chemical Analysis of MI-1-P Synthase and Comparison of These Data to the Protein Predicted from the INOl DNA Sequence-The reported molecular weight of the MI-1-P synthase subunit as estimated by sodium dodecyl sulfate gel


120

100

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FIG. 4. The sequence of 3680 nucleotides of the genomic DNA fragment containing the INOl gene was determined as described under "Materials and Methods." The start of translation of the INOl gene is found at position 1380, and the initiation codon ATG is outlined with a box. Another potential translation start site is simi- larly indicated starting at position 1315. The TATA sequence at position 1258 is likewise underlined.

electrophoresis is 62,000 (9), consistent with the molecular weight of 62,842 predicted from the DNA sequence for the protein encoded by the INOl gene. The amino acid composition of purified MI-1-P synthase was determined as described under "Materials and Methods" and the composition is dis-

GCT ARA GGC G T T AAG CM CCA AAC TAC TIC GGC TCC A X ; ACT WA E T TCT ACC TTG IWI Ala LYS Gly Val LyS G l n Pro Asn Tyr Phe GlY SeZ Met Thr Gln CYS Ser Thr Leu Lyr

CTG GGT ATC GAT GCG GAG GGG AAT GAC G T T TAT GCT CCT TIT AAC TCT CTG TTG CCC ATG

Leu Gly Ile Asp Ala Glu Gly Asn Asp V a l Tyr A l a PTO Phe AS" Ser Leu Leu Pro Met

GTT AGC CCA AAG CAC TIT GTC GTC TCT GGT TGG GAC A X .UT AAC GCA GAT CTA TAC GAR Val Ser Pro Ly9 HIS Phe Val Val ser Gly Trp Asp n e Asn A511 Ala Asp Leu Tyr Glu

GCT ATG CAG AGA AGT CAA GTT CTC GAA TAT GAT CTG uv\ uv\ CGC TTG AAG GCG AAG A X ;

A111 Met Gln Arg Ser Gln Val Leu Glu Tyr Asp Leu Gln Gln Arg Leu LYS A l a LyS Met

TCC TTG GTG AAG CCT CTT CCT TCC ATT TAC TAC CCT GAT TTC ATT GCA GCT ART GAT Ser Leu Val Lys Pro Leu Pro Ser Ile Tyr Tyr Pro Asp Phe Ile Ala Ala As" G l n Asp

GAG AGA GCC AAT CAA TGC ATC AAT TTG GAT GAA ARA GGC AAC GTA ACC ACG AGG GGT AAG G l u Arg Ala Rsn Gln Cys Ile A m Leu Asp G1u Lys Gly A m Val Thr Thr Arq Gly Lys

TGG ACC CAT CTG uv\ CGC ATC AtA CGC GAT A X CAG AAT TTC RRR GAR GAA AAC GCC CTT Trp Thr His Leu Gln Arg Ile Arq Arg Asp Ile Gln A m Phe Lyo G l u Glu A m Ala Leu

GAT AMI GTA ATC GTT CTI TGG ACT GCA ART ACT GAG AGG TAC GTA GAR GTA TCT CCT GGr ASP LYS Val Ile Val Leu Trp Thr A l a Asn Thr G l U Arg Tyr Val Glu V a l Ser Pro Gly

GTT AAT GAC ACC ATG GAA ARC CTC TTG CAG TCT A I T RAG AAT GAC CAT GAA GAG ATT GCT Val ASn Asp Thr Met G l u Asn Leu Leu Gln Ser Ile LYS A m ASP HIS Glu Glu I le A l a

CCT TCC ACG ATC TI7 GCA G C A GCA TCT ATC Tn GAR GGT GTC CCC TAT ATT AAT GGT TCA Pro SeT Thr Ile Phe A l a All Ala Ser Ile Leu Glu Gly Val Pro Tyr Ile Asn Gly Ser

CCG CAG AAT ACT m GTT CCC GGC TTG GTT CAG CTG GCT GAG CAT GAG GGT ACA TTC ATT

Pro Gln A m Thr Phe Val Pro Gly Leu Val Gln Leu Ala Glu His Glu Gly Thr Phe Ile

GCG Gw\ GAC GAT CTC AAG TCG GGA CAA ACC AAG TTG AAG TCT GT? CTG GCC CAG TTC TTA

Ala Gly ASP ASP Leu Lys Ser Gly Gln Thr LYS Leu LYP Ser V a l Leu A l a Gln Phe Leu

GTG GAT GCA GGT ATT RRR CCG G K TCC ATT GCA TCC TAT AAC CAT TTA GGC AAT AAT GAC Val ASP AI^ Gly n e LYS pro ve.1 ser n e la ser q r AS" HIS L ~ Y GIY AS^ AS" ASP

GGT TAT AAC TTA TCT GCT CCA ARA CAA TIT AGG TCT AAG GAG A T I TCC RRR AGT TCT G X Gly Tyr A4n Leu Ser Ala Pro Lys G l n Phe Arg Ser Lys Glu Ile Ser Lys Ser Ser Val

ATA GAT GAC ATC ATC GCG TCT AAT GAT ATC I l G TAC AAT GAT RRR CTG GGT AMI AMI GTT 11e ASP ASP 11e 11e la ser AS" ASP n e Leu q r Asn ASP LYS ~ e u Gly LYS LYO va1

GAC CAC TGC A X GTC ATC ARA TAT ATG RAG CCC GTC GGG GAC T U RRR GTG GCA ATG GAC ~ s p HIS cys 11e val n e LYS ~ y r Met LYS pro Val Gly ASP Ser LYS Val la Met ASP

GAG TAT TAC AGT GAG T T G ATG TTA GGT GGC CAT AAC CGG A T T TCC ATT CAC AAT G T T TGC G ~ U ~ y r T Y ~ ser GI" leu Met ~ e u Gly ~ l y HIP A m A r g Ile ser Ile HIS ~ s n val cyo

GAA GAT TCT TTA CTG GCT ACC GCC TPG ATC ATC GAT CTT TTA GN ATG acT GAG m TGT GI" ASP ser MU ~ e u la ~ h r A L ~ L ~ Y n e Ile ASP M U M U va1 Het Rlr ~ l u Phe cyo

ACA RGA GTG TCC TAT AAG AAG GTG GAC CCA GTT ARA GAA GAT GCT GGC RRR Tl'C GAR GAA Thr A r g Val Ser Tyyr Lys Lys V a l Asp Pro V a l Lys Glu Asp Ala Gly Lys Phe Glu G l u

CTT TTA TCC AGT TIT ARC CTT CTT GAG TTA CTG GTT ARA AGC TCC ATT AAC R A G AAC CAG

Leu Leu Ser Ser Phe A m Leu Leu Glu Leu Leu Val LyS Ser Ser Ile Asn Lys Asn Gin

GAT TTA CAC CCG GTG AAT GGC TTA AAC AAG CAA AGA ACC GCC TTA GAR AAT m TTA AGA Asp leu HIS Pro Val Aon Gly Leu Rsn Lys Gln Arq Thr Ala Leu Glu Asn Phe Leu Arg

TTG TTG ATT GGA TTG CCT TCT CAA AMI CGA ACT AAG A l l CGA AWL GM; A T T GTT GTA A X Leu Leu Ile Gly Leu Pro Ser Gln Lys Iug Thr Lys Ile Arq Arq Glu Ile V a l Val Ile

TCA TlT uv\ CGA CTC TCT TCT TlT K C GCC TAC CTA T M U A liRc AAG ACA TTC ACC Ser Phe Gln Arq Leu Ser Phe Ser Phe Ser Ala Tyr MU

ATT ATC CTA TTA TCC CTT CCA TCA ATA CAT ATA CTT Iv\c ATA ACG TIT ITA AAT AAC TAT

uLI\ CCA GTC TIT ATA TTl m m m CTT T T G AAC TAT TGC CTC m G X ACT K G Tl'C

TTA AAG GGG CGT m TTA lTT TIT TIT TIT m TTC ACT TGA GGA GAG CGA GAA AGT GCT

GTA TlT ATT CAA GGG CCA CCT CAG TAA AGA GAA GAR A?& AGA GAA ASA RRR AMI GAA GGT

GGT GTA ATG TGC GAC CAC TTC AAC AAG GCC CAG TGA am TAA TAT ATA ACG AMI AGG AGG

AGG ACG AGA AGA AGA AGC T W RRR CAC M T GCG TAT GCG tAC T U CCA GM AAT KG AX

GCT GTT ATC AAG CAG C W ACC Gu CTA A X ; AAC CU: nir CTA TCT CM CAC AMI CTA AX;

GAR TCA TTA CAG CAT ACT TCC ATA A X ; TE ACC GM TE AGA AAT CCA MA CTT A X GCC

GAR GAR ATA CTA C W ACT TTA TGT CAT TAT TSr CGA ClC ATT GAC T M TCT A X TAA CGT

ACC TCA TAG AM AAC CAT CCT CAA RIT W CAC TTA CCT GAT CTT TAT GM; T T G GTT CM

TAT ACC VI? AAC GTG GTA CC

FIG. 4"continued

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1278 Primary Structure of Yeast myo-Inositol-1 -phosphate Synthase

played in Table 11. A comparison of the amino acid composition generated from the DNA sequence of the large open reading frame corresponding to the INOl gene with the chemically determined amino acid composition of MI-1-P synthase (Table 111) reveals an excellent correlation between the two.

The enzyme subunit was subjected to amino-terminal analysis as described under “Materials and Methods,” and the results are displayed in Table IV. The amino terminus of the predicted protein (Fig. 4) (Met-Thr-Glu-Asp-Asn-Ile-Ala- Pro) when compared to the chemically determined amino terminus of MI-1-P synthase (Table IV) (Met-(Ser/Thr)-Glu- Asp-Asn-Ile-Ala-Pro) provides confirmation that the open reading frame starting at position 1380 (Fig. 4) of the INOl gene encodes MI-1-P synthase.

Bionet’s computer program was used to search the NBRF protein database for homologies to the INOl coding sequence. No significant homologies to any reported protein sequences

Peptides I NO 1 gene

U - 1Kb -c

FIG. 5. Computer analysis of the DNA fragment containing the INOl gene shows that there are three potential peptides which could be read in the same reading frame as the largest open reading frame, corresponding to the INOl gene.

were detected in the coding region of the INOl gene. Table V lists sequences in the 5‘- and 3”noncoding regions of the INOl gene which are similar to conserved sequences which have been reported adjacent to the coding regions of other eukaryotic genes.

An Analysis of the DNA Sequence of the Open Reading Frame Adjacent to INOl-An analysis of the second largest open reading frame, encoded in the sequenced DNA adjacent to INOl (Fig. 5), shows that it has features that could function in the insertion or transfer of the protein across a membrane. The open reading frame for this peptide begins at position 542 (Fig. 4). The first 21 amino acids contain charged and uncharged residues (net charge = +2) and are followed by 16 hydrophobic residues which are in turn followed by 3 charged residues (Lys, Arg, Lys). The structure of this amino-terminal region resembles that of known signal sequences (Table VI). The predicted peptide consists of 133 amino acids and has a molecular weight of 15,182. The computer-generated hydrophilicity plot (Fig. 7) shows that the hydrophobic pockets occur near the amino terminus of the putative protein.

Construction and Analysis of Strains Bearing Gene Disrup- tions of INOl and the Adjacent Open Reading Frame-Gene disruptions were constructed in the chromosomal locus corresponding to the cloned INOl gene and the 133-amino acid adjacent open reading frame. The gene disruptions were constructed in diploid strain DC5D (Table I) as described under “Materials and Methods” and illustrated in Fig. 3. The diploid strains heterozygous for the gene disruptions were sporulated and subjected to tetrad analysis. The diploid strain bearing the disruption of the large open reading frame corresponding to the INOl gene segregated Ino’/Ino- and Leu+/Leu- phenotypes at the expected tetrad ratio of 2+:2-. The spores which were Ino+ in phenotype were all Leu- and the Leu+ spores were all Ino- in phenotype confirming, on a genetic level, replacement of the INOl gene with the LEU2 gene. Southern blot analysis confirmed the gene replacement at a molecular level (Fig. 8). The inositol-requiring (Ino-) spores, derived from sporulation of the diploid bearing the INOl disruption, were subjected to genetic complementation analysis. Haploid strains of genotype inol-13 (Table I) (inol-13 is

TABLE I1 Codon usage and amino acid composition predicted from the DNA sequence of INOI, the structural gene for

MI-I-P synthase Codon usaee

AAA ( L y s ) : 21 A C A ( T h r ) : 5 A G A ( A r g ) : 8 A T A ( I 1 e ) : 2 C A A ( G 1 n ) : 17 C C A ( P r o ) : 5 C G A ( A r g ) : 4 CTA ( L e u ) : 2 G A A ( G 1 u ) : 17 GCA ( A l a ) : 9 G G A ( G 1 y ) : 3 G T A ( V a 1 ) : 7 T A A ( S T 0 P ) : 1 T C A ( S e r ) : 3 T G A ( S T 0 P ) : 0 T T A ( L e u ) : 14

A A C ( A s n ) : 14 A C C ( T h r ) : 10 A G C ( S e r ) : 4 A T C ( I 1 e ) : 16 C A C ( H i s ) : 4 C C C ( P r o ) : 5 CGC ( A r g ) : 4 CTC ( L e u ) : 6 G A C ( A s p ) : 15 GCC ( A l a ) : 6 GGC ( G l y ) : 9 G T C ( V a 1 ) : 8 T A C ( T y r ) : 12 T C C ( S e r ) : 13 T G C ( C y s ) : 4 TTC ( P h e ) : 10

A A G ( L y s ) : 23 A C G ( T h r ) : 5 AGG ( A r g ) : 5 A T G ( M e t ) : 11 C A G ( G 1 n ) : 7 C C G ( P r o ) : 4 CGG ( A r g ) : 2 C T G ( L e u ) : 10 G A G ( G 1 u ) : 14 G C G ( A 1 a ) : 4 GGG ( G l y ) : 2 G T G ( V a 1 ) : 7 T A G ( S T 0 P ) : 0 TCG ( S e r ) : 1 TGG ( T r p ) : 9 T T G ( L e u ) : 15

A A T ( A s n ) : 21 A C T ( T h r ) : 10 A G T ( S e r ) : 6 A T T ( I 1 e ) : 17 CAT ( H i s ) : 6 C C T ( P r o ) : 8 CGT ( A r g ) : 0 C T T ( L e u ) : 9 G A T ( A s p ) : 19 G C T ( A 1 a ) : 13 G G T ( G 1 y ) : 11 G T T ( V a 1 ) : 19 T A T ( T y r ) : 10 T C T ( S e r ) : 15 T G T ( C y s ) : 2 T T T ( P h e ) : 11

Predicted amino acid comDosition

A l a : 32 (5 .8%) A r g : 23 ( 4 . 2 % ) C y s : 6 ( 1 . 1 % ) G l n : 24 ( 4 . 3 % ) H i s : 10 ( 1 . 8 % ) I l e : 35 ( 6 . 3 % ) Met: 11 (2 .0%) Phe: 21 ( 3 . 8 % ) T h r : 30 ( 5 . 4 % ) Trp : 9 ( 1 . 6 % )

A s n : 35 ( 6 . 3 % ) A s p : 34 (6 .1%) G l u : 31 (5 .6%) G l y : 25 ( 4 . 5 % ) L e u : 56 (10 .1%) L y s : 44 (8 .0%) Pro: 22 ( 4 . 0 % ) Ser: 42 ( 7 . 6 % ) T y r : 22 (4 .0%) V a l : 41 ( 7 . 4 % )


Hydrophilic ...........................................................

+ 2

+ I

+O

- 1

- 2

- 3 7

- 4 , I , I , I I I I I I I I , I , I ( I -

- ........................................................... Hydrophobic

0.0 0.1 0.2 0 3 0.4 0.5 0 6 0.7 0.8 0 9 1.0 Fraction of Length

FIG. 6. Hydrophobicity plot of the INOl gene product as determined by the DNA Inspector I1 program.

TABLE I11 Comparison of the chemically determined amino acid composition of

MI-1-P synthase purified from wild-type yeast with the predicted amino acid composition generated from computer analysis of the

DNA seauence of the INOl gene

of Composition average Amino acid

MI-l-P svnthase chemically determined

Histidine Lysine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Asparagine

Glutamine

1.8 7.8 3.0

*12.7 5.9 6.8

*10.8 5.4 6.8 7.5 0.9 7.6 1.6 5.7 8.7 3.7 4.0

* Value contained in

* Value contained in aspartic acid

glutamic acid

predicted from DNA Composition

sequence analysis

% total nmol 1.8 8.0 4.2 6.1 5.4 7.6 5.6 4.0 4.5 5.8 1.1 7.4 2.0 6.3

10.1 4.0 3.8 1.6 6.3

4.3

TABLE IV Primary sequence of the amino terminus of MI-1-P synthase

Amino-terminal sequence was determined using MI-1-P synthase subunit purified as described under “Materials and Methods.” The two high performance liquid chromatography (HPLC) systems are described under “Materials and Methods.”

Residue Amino acid Method of detection

HPLC I HPLC I1

1 Met X X 24 Ser/Thr X X 3 Glu X X 4 ASP x X 5 Asn X X 6 Ile X X 7 Ala X X 8 Pro X X

Insufficient resolution of serine and threonine residues was obtained to permit positive identification.

a missense allele derived from mutagenesis of a wild-type strain with ethylmethane sulfonate (10)) were crossed to Ino- strains carrying the INOl gene disruption. Diploid strains produced from such crosses were inositol auxotrophs. Thus, the INOl gene disruption failed to complement existing in01 alleles, providing additional genetic confirmation of the iden- tity of the large open reading frame as the INOl locus.

Cellular extracts made from haploid strains carrying the INOl gene disruption were subjected to Western blot analysis (39). Such strains failed to express any material cross-reactive to MI-1-P synthase antibody (Fig. 9). In comparison, the control strains, both wild-type and a haploid strain bearing the inol-13 allele (inol-13 strains produce inactive MI-1-P synthase) did express material cross-reactive to anti-MI-1-P synthase. As expected, the regulatory mutant ino4, which fails to derepress the enzyme subunit (9), does not express material cross-reactive to MI-1-P synthase.

The largest of the small open reading frames adjacent to INOl was also disrupted. The analysis was carried out in a fashion similar to that described above for the INOl disruption. Haploid strains carrying the disruption of the small open reading frame were found to be viable. They were not auxotrophic for inositol and had no evident growth defect. North- ern blot analysis (41) confirmed the absence of the transcript of the small open reading frame (Fig. 10) in haploid cells carrying the disruption. Strains carrying the disruption of the small open reading frame were shown to excrete inositol by a bioassay (14). The excretion phenotype was found to be genetically cis-dominant when diploids heterozygous for the disruption were constructed. However, Western blot analysis (Fig. 11) of MI-1-P synthase expression in haploid strains carrying the disruption of the small open reading frame revealed that MI-1-P synthase expression was repressed nor- mally in cells grown in the presence of inositol. Thus, disruption of the small open reading frame led to excretion of inositol but did not appear to affect regulation of MI-1-P synthase.

The phospholipid composition of both categories of gene disruption mutants was analyzed. No significant differences in the phospholipid compositions of the gene disruptants were found when compared to the wild-type composition (data not shown).

DISCUSSION

Inositol has recently emerged as a biologically significant molecule in many areas of animal, plant, and yeast cell physiology (1). The availability of the mutants of inositol biosynthesis in S. cereuisiae provides opportunity for a genetic and molecular dissection of this important eukaryotic pathway.

This study has provided, for the first time, a direct comparison of the chemically determined amino acid composition of MI-1-P synthase with that determined by molecular analysis. This comparison shows excellent agreement of the two compositions providing direct substantiation that INOl is the structural gene for MI-1-P synthase. The molecular weight of the protein predicted from the DNA sequences is essentially identical to the molecular weight reported previously for the subunit of MI-1-P synthase (9). The analysis of eight amino acids from the amino terminus of the protein (Table IV) established the reading frame and translational start at position 1380. The amino-terminal analysis was particularly important in the case of the INOl gene since the computer analysis of the large open reading frame revealed a potentially larger protein generated using the AUG codon at position 1315 (Fig. 4). The finding that translation starts at position

1280 Primary St ruc ture of Yeast myo-Inositol-1 -phosphate Synthase TABLE V

Conserved sequences in the flanking regions of the INOl gene 5' Promoter region

Consensus sequence

INOl equivalent

Distance

ATG from Function Reference

GGPyCAAG GGCCAAG -274 Modulator 43-45

CAAT CAAT -140 Yeast promoter 43 TATA TATAAATT -122 mRNAselectionsite 43, 15. 46

TATTTAAT -247

CTAAATT -70 PumTGPuXT ACAAAACA -3 T r a n s l a t i o n a l s t a r t s i t e 47

3' Termination region

Consensus sequence for TAA eukaryoticgenes TAG 140basepa i r s ( T - r i c h ) TAG TAGT ( A T - r i c h ) TIT (48) TGA TATGT

INOl e q u i v a l e n t TAA 60basepa i r s ( T - r i c h ) TAGT ( A T - r i c h )

TABLE VI Proteins with some homology to the amino terminus of the protein predicted from the 133-amino acid open reading

frame adjacent to the IN01 gene Protein Amino-terminal sequence

Small openreading frame

Pancreat ic secretoryproteins (49)

GeneEproteinof OX-174 (50)

B n L F l p r o t e i n o f Eps te in-Barrv i rus (51)

Gag p o l y p r o t e i n o f t h e fe l inesarcomavi rus ( 5 2 )

Thr-Leu-Leu-Asn-Leu-Leu-Leu-Phe-Leu-Leu Leu-Phe-Phe-Pro-Ala-Ile-Ile Ala-Leu-Leu-Leu-Leu-Leu-Leu-Ala Leu-Leu-Leu-Ala-Tyr-Val-Ala-Phe Thr-Leu-Ala-Phe-Leu-Leu-Leu-Leu-Ser Leu-Leu-Leu-Leu-Ser-Leu-Leu-Ile Ser-Leu-Gly-Leu-Ala-Leu-Leu-Leu-Leu Leu-Ala-Leu-Leu-Phe-Trp-Leu Gln-Leu-Len-Gln-Ala-Leu-Leu-Thr-Gly-Glu Glu-Arg-Gln-Arg-Val-Leu-Leu

+ 4 - + 3 - ...........................................................

Hydrophilic

- + 2 4 I

1 2 3 4 5 6

J

- 3 - - ...........................................................

- 4 1 , I , , , I , I , I , Hydrophobic

Fraction of Length

I I I ' I ' 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

FIG. 7. Hydropkylicity plot of the largest open reading frame upstream of the INOZ gene shows that it has features of a membrane-associated protein.

1380 is also consistent with the finding by Hirsch (57) that the major INOl transcript starts five nucleotides upstream from the start of translation (i.e. a t position 1375 relative to Fig. 4). Codon usage analysis was consistent with the identification of a moderately expressed yeast protein (41,42).

The hydrophilicity analysis of MI-1-P synthase illustrated in Fig. 6 has confirmed that MI-1-P synthase, as expected for a cytoplasmic enzyme, has no extensive regions of hydrophobicity. Initially, the enzyme was purified using its affinity to hydrophobic resins (9), suggesting that i t might be unusually hydrophobic for a cytoplasmic enzyme. However, the absence of extensive stretches of hydrophobic amino acids in the MI-

FIG. 8. Southern blot analysis (38) was used to locate the LEU2 gene at the INOI locus. Yeast genomic DNA was cut with restriction enzyme Hind111 which generates the INOl gene on a 6.3- kb fragment (lower arrow). The insertion of the LEU2 gene into the INOl region (as illustrated in Fig. 3) generated a fragment of approximately 7.9 kb (upper arrow). To demonstrate the genetic linkage of the leucine prototrophy to the INOI locus, a transformed diploid was sporulated and 20 tetrads were analyzed. The leucine prototrophy in the meiotic products is genetically linked to the I N 0 1 locus and co- segregates with the larger restriction fragment produced by the gene disruption. That is, the Ino- phenotype co-segregated with the Leu+ phenotype in all tetrads. Lanes I and 6 contain DNA isolated from the disrupted diploid. These strains possess both restriction fragments. Lanes 2 and 3 contain DNA from spores that are phenotypi- cally Leu- (smaller restriction fragment), while lanes 4 and 5 contain DNA isolated from Leu' spores (larger restriction fragment produced by gene disruption).

1-P synthase sequence suggests that the protein is probably not associated with membrane. The determination of the primary sequence of MI-1-P synthase, however, opens the possibility of more detailed studies in the future of the structure of the enzyme and may permit localization of active sites within the primary sequence. This enzyme is known to cata- lyze a complex series of reactions that involve a t least three


c

I 2 3 4 5 6 7 8 FIG. 9. Western blot analysis (39) was used to detect the

presence or absence of the IN01 gene product (MI-1-P synthase) in the gene disruptants. Lane 1 contains a cell extract of the wild-type haploid DC5. Lane 2 is empty. Lane 3 contains an extract of inol-13 mutant cells. (The inol-I3 strain bears a missense mutation that makes an inactive protein which is immunologically cross-reactive (9).) Lane 4 contains a cell extract of a strain bearing the in04 regulatory mutation. (The in04 mutants are unable to derepress MI-1-P synthase and therefore produce no detectable cross- reactive material (9).) Lanes 5-8 contain extracts of colonies grown from the meiotic products of the diploid analyzed in Figure 8. The phenotypes of the spores in lanes 5 and 6 are Ino+, Leu-, while the ones in lanes 7 and 8 are Ino-, Leu+. (In other words, lanes 7 and 8 contain extracts from strains bearing the disruption of the INOl sequence.) Cells of all strains were grown in the presence of 10 pM inositol, a level of inositol sufficient to permit partial derepression of MI-1-P synthase (9, 16) while supporting growth of inositol auxotrophs. The arrow marks the position of the molecular weight 62,000 MI-1-P synthase subunit.

I 2 3 4 FIG. 10. Northern blot analysis of strains bearing the dis-

ruption of the small open reading frame. RNA was extracted from cells grown from the four spore colonies of a single tetrad dissected from the diploid strain bearing the gene disruption. Cells were grown in the absence of inositol. Lane 1, spore colony A, carrying the disruption (Leu+). Lane 2, spore colony B (Leu-) no disruption. Lane 3, spore colony C (Leu-) no disruption. hne 4, spore colony D carrying the disruption (Leu'). Equal amounts of total RNA were applied to each lane. The probe used spanned the entire INOl region (Fig. 2). Note the presence of the INOl transcript (upper arrow) in all four lanes. The smaller transcript (lower arrow) is missing in the strains A and D carrying the disruption (lanes 1 and 4).

partial reactions (5-7). It is not known how many active sites there are within MI-1-P synthase nor how they are positioned relative to one another. Future analysis of the positioning of the in01 mutations that destroy catalytic activity of the enzyme relative to the primary sequence may permit these questions to be addressed.

Comparison of the primary sequence of the INOl gene product MI-1-P synthase to proteins found in Bionet's data- bank revealed no homology to any other previously analyzed protein. It will be interesting in the future to compare the structure of the enzyme from yeast to other proteins which utilize glucose 6-phosphate as substrate or NADH as cofactor.

- 1 2 3 4 5

FIG. 11. Analysis of regulation of MI-1-P synthase in cells bearing the disruption of the small open reading frame. West- ern blot analysis was used to detect the presence of the MI-1-P synthase subunit (arrow). Lane I , extract of cells from spore colony A (Fig. 10) bearing the disruption (Leu+) of the small open reading frame. Cells were grown in the presence of 75 pM inositol. Lane 2, extract of cells of spore colony A bearing the gene disruption grown in the absence of inositol. Lane 3, extract of cells of the ind strain which fails to derepress the MI-1-P synthase subunit; grown in 10 p M inositol. Lane 4, extract of cells of spore colony D (Fig. 10) (Leu') carrying the gene disruption; grown in the absence of inositol. Lane 5, extract of cells of spore colony D grown in the presence of 75 pM inositol. The absence of the MI-1-P synthase subunit in cells of the Leu' strains (spore colonies A and D; Fig. 10) grown in the presence of 75 p~ inositol (lanes I and 5) demonstrates that MI-1-P synthase is fully repressible when the small open reading frame is disrupted.

Since MI-1-P synthase from other sources (6) is of similar size and has the same cofactor requirement, it will be interesting to determine whether the primary sequence of MI-1-P synthase has been conserved in evolution. Experiments are presently underway using the yeast INOl gene and an antibody specific to its gene product MI-1-P synthase to isolate the analogous gene from higher plants.

Table V summarizes the location, putative function, and similarity of sequences in and around the INOl gene as compared to conserved sequences found in the regulatory regions of other genes whose sequences have been published. TATA-like sequences are present a t positions -122 and -247 relative to the start of translation in the 5' region of the INOl gene. (These positions correspond to nucleotides 1193 and 1258 on Fig. 4). These sequences resemble the TATA box sequence that is present in most eukaryotic genes and is implicated in the eukaryotic RNA polymerase recognition site (43). Another sequence thought to be important for transcription initiation in eukaryotes is the CAAT sequence (43). In the INOl gene, this sequence appears at position -140 relative to the start of translation (i.e. nucleotide 1240 in Fig. 4).

The 133-amino acid open reading frame adjacent to INOl has been shown to encode a mRNA species of 0.6 kb (16). The region 5' to the first ATG of this open reading frame is filled with TATA-like sequences. For example, TATAATT is located at position 529 (Fig. 4), TATATAAA at position 431, and TATATA at position 250. A potential poly(A) addition site (AAATAA) is located a t position 431. Also, the sequence AGCCAGCTGCAG that is located at position 465 has been identified from mammalian sources as a phorbol ester-binding site (53). Further analysis will be required before the function(s) of these sequences can be determined. However, the putative peptide encoded by this open reading frame has an intriguing structure. The hydrophilicity plot shown in Fig. 7 indicates that there are long stretches of hydrophobic residues that could be readily associated with a membrane. Although a rigorous determination of the transmembrane disposition of a protein generally requires the use of macromolecular probes such as antibodies, proteases, and nonpenetrating or selective labeling reagents, the availability of complete primary sequences for numerous membrane proteins has allowed tenta- tive predictions of their transmembrane disposition to be made on the basis of hydropathy plots (54). Search of the NBRF protein database for proteins homologous to the 133- amino acid open reading frame indicated that it shares ho-

1282 Primary Structure of Yeast myo-Inositol-1 -phosphate Synthase

mology with some membrane-associated proteins found in the data bank (Table VI). In fact, the hydrophobic stretch of amino acid residues starting at position 605 following the putative signal sequence is quite homologous to a sequence in the amino terminus of rat liver cytochrome P-450 PB-4 (55). The sequence (Met-Glu-Pro-(Ser)-Ile-Leu-Leu-Leu-Leu-Ala- Leu-Leu-Val-Gly-Phe-Leu-Leu-Leu-Leu-Val) of rat liver cytochrome P-450 PB-4 has been shown to anchor the protein to the endosplasmic reticulum (55). The hydrophobic region in the amino-terminal region of the 133-amino acid open reading frame also appears to share homologies to similar regions found in the hypothetical BNLFl protein of Epstein- Barr virus, the gag polyprotein of the feline sarcoma virus, the gene E protein of 9x174, and the amino-terminal sequence of precursors to pancreatic secretory proteins (Table VI).

Gene disruptions designed to eliminate, individually, expression of the open reading frame upstream of the ZNOl gene and the IN01 gene itself proved to be viable. Unlike most yeast loci, but similar to the yeast HIS1 locus, the IN01 locus lacks allelic representatives that are suppressible by known nonsense suppressors (56). This finding suggested that premature termination of translation of the IN01 gene product MI-1-P synthase might be incompatible with cell viability. Testing of this hypothesis was a major motivation for creating null mutations at ZNOl. However, genetic complementation analysis as well as Western blot analysis confirmed the exist- ence of viable null mutations at INOl. The null mutants are simply inositol auxotrophs similar in growth and phospholipid composition to other inol alleles. Thus, failure to detect nonsense mutants at the IN01 locus remains an unexplained phenomenon.

The disruption of the small open reading frame upstream of the ZNOl gene is also viable and causes the cell to excrete inositol, a phenotype which is associated with mutants defec- tive in regulation of phospholipid biosynthesis (13, 14). The excretion phenotype is genetically cis-dominant in the strain disrupted in the small open reading frame, whereas most other Opi- mutants are recessive. However, unlike other regulatory mutants with the inositol excretion phenotype such as opil (13), synthesis of MI-1-P synthase is repressed in the presence of inositol in cells carrying the gene disruption. The insertion of the LEU2 gene in the region adjacent to the ZNOl gene may have affected INOl transcription, leading to over- production of inositol. At present, we believe that this is a probable explanation for the inositol excretion phenotype observed in strains bearing the disruption of the small open reading frame. Because the disruption of the small open reading frame had no evident effect upon growth, it may be concluded that it does not encode a protein essential for growth under the laboratory conditions described in this report. No further conclusion can be drawn at this time about the function of its gene product in phospholipid biosynthesis or regulation or in other cellular processes. Future experiments designed to identify the gene product (gene fusions, antibody production) would give a better understanding of the role of the product of this gene in yeast cell biology.

Acknowledgments-John Hill and Pat McGraw provided much helpful discussion as well as critical reading of the manuscript. We are indebted to Howard Steinman for his assistance in determining the amino acid composition of MI-1-P synthase and to Edith Palmieri for preparation of oligonucleotides.

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Documents

Biosynthesis of Inositol in Yeast · variety of neurotransmitters, hormones, and growth factors (for review, see Ref. 1). In the yeast Saccharomyces cereuisiae, synthesis of the phospholipid