Henry (9). MI-1-P synthase purified by column chromatography was assayed by the rapid ...... Majerus, P. W., Connolly, T. M., Deckmyn, H., Ross, T. S., Bross,.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Q 1989 by The American Society for Biochemistry andMolecular 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 ANALYSIS OF ITS STRUCTURALGENE, THE INOl LOCUS*
5.5.1.4)AND
FUNCTIONAL
(Received for publication, June 6, 1988)
Margaret Dean-Johnson$ and SusanA. 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 variety of neurotransmitters, hormones, and growth factors Saccharomyces cerevisiaeI N O l gene and its product, (for review, see Ref. 1). L-myo-inositol-1-phosphatesynthase (EC 5.6.1.4) has In the yeast Saccharomyces cereuisiae, synthesis of the been carried out. The sequence of the entireI N O l gene phospholipid precursor inositol is one of the most highly and surrounding regions has been determined. Com- regulated aspects of phospholipid metabolism. Regulation puter analysis of the DNA sequence revealed four po- centers on the cytoplasmic enzyme MI-1-P synthase.’ MI-1tential peptides. The largestopen reading frameof 553 P synthase catalyzes the synthesis of inositol 1-phosphate de amino acids predicted a peptide with a molecular novo via the cyclization of glucose 6-phosphate (2-4). While weight of 62,842. The amino acid composition and the precise reaction mechanism has not been elucidated, the amino terminus of purified L-myo-inositol-l-phosphate synthase were chemically determined and com- absolute requirement for NAD with no net gain in NADH (5) pared to the amino acid composition and amino termi- suggests that theoverall reaction consists of a tightlycoupled nus of the proteinpredicted from the DNA sequence of oxidation and reduction. Two intermediates, 5-ketoglucose 6the largeopen reading frame. This analysis established phosphate andinosose 2,1-phosphate, have been proposed (5been directly identified and each is assumed that the largeopen reading frame encodes L-myo-ino- 7), but neither has to be tightly bound to theenzyme (5, 8). sitol-1-phosphate synthase. Thelargest of several The molecular weight of the native enzyme in yeast as I N O l predicted small open reading frames adjacent to a protein of 133 amino acids with a molecular weight determined by gel filtration is approximately 240,000 (9). A of 15,182 and features which suggested that the en- single subunit of 62,000 is detected upon sodium dodecyl sulfate gel electrophoresis of the purified enzyme (9). Based coded protein may be membrane-associated. A gene disruption was constructedat I N O l by elim- upon an immunological analysis of inol mutants, the INOl inating a portion of the coding sequence and replacing locus was identified as theprobable structural gene encoding it with another sequence. Strains carrying the gene the MI-1-P synthase subunit in yeast(9). disruption failed to express any protein cross-reactive In yeast, MI-1-P synthase is regulated both in response to to antibody directedagainst L-myo-inositol-l-phos- exogenous inositol and to unlinked regulatory genes (9-16). phatesynthase. Although auxotrophicfor inositol, The addition of inositol to logarithmically growing cultures strains carrying thegene disruption were completely results in a 50-fold, time-dependent decrease in the enzyme’s viable when supplemented with inositol. In a similar activity. Immunological and biochemical evidence supports fashion, a gene disruption was constructed in the chrothe conclusion that the pathway for inositol biosynthesis in mosomallocus of the 133-amino acid open reading yeast is regulated by the repression of enzyme synthesis (9). frame. This mutation did not affect viability but did The S. cereuisiae INOl gene, which encodes MI-1-P synthase, cause inositol to be excreted from thecell. 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 Recently, studies which encompass many aspects of animal, expression of the gene under conditions known to regulate plant, and yeast cell physiology have shown that the biosynphospholipid synthesis (16). RNA blot hybridization detected thesis and metabolism of inositol and the inositol phosphotwo RNA species of 1.8 and 0.6 kb (16). Complementation lipids play central roles in signal transmission for a wide analysis showed that the 1.8-kb RNA encodes the IN01 gene * This work was supported in part by Grant GM-19629 (to S. A. product (16). The level of RNA was repressed 30-fold when H.) from the National Institutes of Health. This report was taken in the cells were grown in the presence of inositol and choline part from a Ph.D. thesis submitted in 1987 by M. D. J. to the Albert together (16). The level of the 0.6-kb RNA is affected to a Einstein College of Medicine. The costs of publication of this article lesser degree bymany of the same factors that influence INOl were defrayed in part by the payment of page charges. This article expression (16). A similar pattern of regulation has been must therefore be hereby marked “aduertisement” in accordance with shown for other enzymes of phospholipid biosynthesis in S. 18 U.S.C. Section 1734 solelyto indicate this fact. The nucleotide sequence(s)reported in this paper has been submitted cereuisiae (17-22) (Fig. 1). Mutations at loci unlinked to INOl are also known to affect to the GenBankTM/EMBL Data Bank withaccessionnumber(s) expression of MI-1-P synthase. The opil mutant, identified 5044.53. $ Supported by National Institutes of Health Training Grant GM- on the basis of a bioassay for inositol excretion (141, causes 07128. Present address: Dept. of Biology, Yale University, New Ha- the MI-1-Psynthasesubunit to be constitutively overexven, CT 06520. Present address: Dept. of Biological Sciences, Carnegie Mellon University, 4400 Fifth Ave., Pittsburgh, PA 15213. To whom reprint requests should be addressed.
The abbreviations used are: MI-1-P synthase, L-myo-inositol-lphosphate synthase; kb, kilobase(s).
1274
Primary Structure
of Yeast myo-Inositol-1
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 havepleiotropic 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. Phospholipidbiosynthesis 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 methylation of phosphatidylethanolamine (PE) in S. cereuisiaemembranes. Kennedy and Weiss (28) described the formation of phosphatidylethanolamine, phosphatidylcholine, phosphatidylmonomethylethanolamine (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), theformation 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 1phosphate.
-phosphate Synthase
1275
complex regulatory network involved in the coordinate regulation of phospholipid biosynthesis in yeast (Fig. 1). MATERIALS ANDMETHODS
Yeast and Bacterial Strains-Yeast strains which wereused for the purification of MI-1-P synthase, preparationof 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 roomagainst 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, 62,000subunit was reisolated from the gel (32). The amino terminus of the protein isolated from the gel wasanalyzed by automated Edmandegradation. 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-aminoacids. 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-ElmerSeries 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 thesequence level. Neither separation system provided sufficient resolution of serine and threonine derivatives 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]orthophosphateand extracted as described previously (15, 18).Phospholipids were separated chromatographically 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-
TABLEI 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. Source or reference
Use
MATa, d e 5
Culbertson and Henry (10)
opil (Over-producer of inositol)
MATa, opil
Greenberg et al. (13, 14)
W303-1A
MATa, leu2,-3112, trpl1, canl-100, u r d - I , ade 2-1, his3-11,15 Mat leu2-3,112, his 3 MATa, leu 2-3,112, inol13 MATaIMATa, leu23.1 12/leu2-3, 112, his3/+, +lade2
Dr. Rodney Rothstein
Isolation of MI-1-P synthase Isolation of MI-1-P synthase Isolation of yeast RNA
Strains
Yeast d e 5 (Wild-type)
DC5 MC13 DC5D Bacterial HblOl
JA300
Genotype
F-, hsdS20 (TB-, mB-), recA13, ara-14, leuB6, proA2, lac Y I ,galK2, rpsL20 (Sm*),XY"~, mtl -I, supE44, XF-,thi-I, leuB6, supE44
Dr. James Broach Klig and Henry (12) Dr. Patricia McGraw
Gene disruption Complementation analysis Gene disruption
Laboratory strain
Isolation of plasmid DNA
Laboratory strain
Isolation of plasmids used for gene disruptions
Primary Structure of Yeast myo-Inositol-1 -phosphate Synthase
1276
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 TAA TATAAA gene, the smaller adjacent open reading frame, and surrounding regions. Arrows indicate the subclone, direction, and thestrategy 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 pH7.0. 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 (37). Fig. 3 outlines construction of the plasmids used for gene disruptions. The method of Southern (38) was used to locate the LEU2 gene at theIN01 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 tetradswere subjected to Western blot (39) analysis to assess the expression of MI-1-Psynthase 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, >
,
1
J
TAG
TAT TGA Smolt ORF 420 bp
I
IN01 gene
1650 bp
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 wasused to disrupt the INOl gene. The 2.2-kb LEU2 gene wasinserted into theplasmid 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 release to the fragment containing the LEU2gene 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. Fragments 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 themethod of Rothstein (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 Asp 16 301 307 CK c" corresponds to the INOl gene (data to support this identifi301 301 31918 Asp 10 cation will bepresented below). Three additionalsmaller open reading frames were detected in theadjacent DNA (Fig. 5). del 68 sst-Bgl Asp 24 " c " The nucleotide sequence of the DNA fragment containing SK 301 del 38 Asp 15 the INOlgene begins 1380 bases upstream of the largest open 1 2 reading frame (Fig. 4). The peptide predicted from the se307 Asp 100 301 2 quence of the large open reading frame consists of 553 amino acids. There isa larger open reading frame starting at position 306 del de144 1 PK 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 307 18 inositol 1-phosphate synthase confirmed that position 1380 RV 5 PB 20 corresponds to the start of translation of inositol 1-phosphate " synthase. Consequently, all subsequent analysis of the sePB 21 RV 29 " quence was performed on the basis of the protein starting at PB 117 RV 21 position 1380. The codonusage in the INOl gene and its " predicted amino acid composition are shown in Table 11. The PB 102 computer analysis revealed that thecodon usage wasconsistent with a moderately expressed yeast protein (41,42). At pH PB 106 7.0, theINOl proteinhas a charge of +2. A computer-1Kb generated hydrophilicity plot for the INOl gene product is FIG. 2. Sequencing strategy. Restriction fragments derived shown in Fig. 6. from plasmids YEpINOl and YIpINOl(12)were cloned into plasmids Chemical Analysis of MI-1-P Synthase and Comparison of pUClS and pUC19 (36). The relevant restriction sites of the yeast sequences in YIpINOl DNA are shown at the top. The arrows and These Data to the Protein Predicted from the INOl DNA names above them indicate the subclone, the direction, and extent of Sequence-The reported molecular weight of the MI-1-Psynthasesubunitasestimated by sodium dodecyl sulfate gel sequence information obtained.
-
-
-
~
-
~
I
-
1277
Primary Structureof Yeast myo-Inositol-1 -phosphate Synthase 60
GCT ARA GGC G T T AAG CM CCA AAC TACTIC GGC TCC A X ; ACT W A E T TCT ACC TTG IWI A l a LYS Gly Val LyS G l n Pro Asn Tyr Phe GlY SeZ Met Thr Gln CYS Ser Thr Leu Lyr
1740
120
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
1000
TIT GTC GTC TCT GGT TGG GAC A X .UT AAC GCA GAT CTA TAC GAR n e Asn A511 Ala Asp Leu Tyr Glu
1060
AGA AGT CAA GTT CTC GAA TAT GAT CTG uv\ uv\CGC TTG AAG GCG AAG A X ; Gln Arg Ser Gln Val Leu Glu Tyr Asp Leu Gln Gln Arg Leu LYS A l a LyS Met
1920
300
TCC TTG GTG AAG CCT CTT CCT TCC A T T TAC TAC CCT GAT TTC A T T GCA GCT ART GAT Ser Leu V a l Lys Pro Leu Pro Ser Ile Tyr Tyr Pro Asp Phe Ile Ala Ala As" G l n Asp
1900
360
GAG
AGA GCC AAT CAA TGC ATC AAT TTG GAT GAA ARA GGC AAC GTA ACC ACG AGG GGT AAG G l u A r g Ala Rsn Gln Cys Ile A m Leu Asp G1u Lys Gly A m Val Thr Thr Arq Gly Lys
2040
420
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 I l e Gln A m Phe Lyo G l u Glu A m Ala Leu
2100
480
GAT AMI GTA ATC GTT CTI TGG ACT GCA ART ACT GAG AGG TAC GTA GAR GTA TCT CCT G Gr ASP LYS Val Ile V a l Leu Trp Thr A l a Asn Thr G l U Arg Tyr Val G l u V a l Ser Pro Gly
2160
GTT AAT GAC ACC ATG GAA ARC
2220
100
GTT AGC CCA AAG CAC
V a l Ser Pro Ly9 HIS Phe V a l Val ser Gly Trp A s p
240
GCT ATG CAG A111 Met
540
CTC TTG CAG TCT A I T RAG AAT GAC CAT GAA GAG ATT GCT
V a l ASn Asp Thr Met G l u Asn Leu Leu G l n Ser Ile LYS A m ASP HIS Glu Glu I l e A l a
600
CCT TCC ACG ATC TI7 GCA G C A GCA TCT ATC T n GAR GGT GTC CCC TAT ATT AAT GGT TCA Pro SeT Thr Ile Phe A l a All Ala Ser Ile Leu G l u Gly Val Pro Tyr Ile Asn Gly Ser
2200
660
CCG CAG AAT ACTm 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 I l e
2340
120
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 G l n Thr LYS Leu LYP Ser V a l Leu A l a Gln Phe Leu
2400
100
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 ~ YGIY AS^ AS" ASP
2460
GGT TAT AAC TTA TCT GCT CCA ARA CAA TIT AGG TCT AAGGAG A T I TCC RRR AGT TCT G X A4n Leu Ser Ala Pro Lys G l n Phe Arg Ser Lys Glu Ile Ser Lys Ser Ser V a l
2520
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
2500
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 v a l n e LYS ~ y r Met LYS pro Val Gly ASP Ser LYS Val la Met ASP
2640
TAC AGT GAG T T G ATG TTA GGT GGC CAT AAC CGG A T T TCC ATT CAC AAT G T T TGC y rT Y ~ ser GI" l e u Met ~ e uGly ~ l yHIP A m A r g Ile ser Ile HIS ~ s val n cyo
2700
GAA GAT TCT TTA CTG GCT ACC GCC TPG ATC ATCGAT CTT TTA GN ATG acT GAG m TGT GI" ASP ser MU ~ e ula ~ h rA L L~ ~ Yn e I l e ASP M U M U va1 Het Rlr ~ l Phe u cyo
2760
ACA
RGA GTG TCC TAT AAGAAG 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
2020
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 G l u Leu Leu Val LyS Ser Ser Ile Asn Lys Asn Gin
2000
CAC CCG GTG AAT GGC TTA AAC AAG CAA AGA ACC GCC TTA GAR AAT m TTA AGA HIS Pro Val Aon G l y Leu Rsn Lys Gln Arq Thr Ala Leu Glu Asn Phe Leu Arg
2940
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 I u g Thr Lys Ile Arq Arq Glu Ile V a l Val Ile
3000
TTC ACC
3060
ATT
ATC CTA TTA TCC CTT CCA TCA ATA CAT ATA CTT Iv\c ATA ACG TIT ITA AAT AAC TAT
3120
uLI\
CCA GTC TIT
ATA
TTl m m m CTT T T G AAC TAT TGC CTC
GGG CGT
m
TTA
040
Gly Tyr 900
960
1020 GAG TAT G ~ U ~
1000
1140
1200
1260 GAT TTA
Asp leu
1320 TTGTTG
1300 CGA CTC TCT TCT TlT K C GCC TAC CTA T M U A liRc Ser Phe Gln Arq Leu Ser Phe Ser Phe Ser Ala Tyr M U TCA TlT uv\
AAG ACA
1440
1500
m
Tl'C
3180
TTC ACT TGA GGA GAG CGA GAA AGT GCT
3240
GTA TlT ATT CAA GGG CCA CCT CAG TAA AGA GAA GAR A?& AGA GAA ASA RRR AMI GAA GGT
3300
GGT GTA ATG TGC GAC CAC TTC AAC AAG GCC CAG TGA am TAA TAT ATA ACG AMI
3360
G X ACT K G
1560 TTA AAG
lTT TIT TIT TIT
m
1620
1600
FIG. 4. The sequence of 3680 nucleotides of the genomicDNA fragment containing the INOl genewas determined as described under "Materials and Methods." The startof translation of the INOl gene is found at position 1380, and the initiation codonATG is outlined with a box. Another potential translation start site is similarly indicated starting at position 1315. The TATA sequence at position 1258 is likewise underlined.
AAT KG AX
3420
GCT GTT ATC AAG CAG C W ACC Gu CTA A X ; AAC CU: nir CTA TCT CM CAC AMI CTA AX;
3400
AGG ACG AGA AGA AGA AGC T W RRR CAC M T GCG
TAT GCG tAC T U CCA
GAR TCA TTA CAG CAT ACT TCC ATA A X ; TE ACC GM
GARGAR
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-
AGG AGG
ATA
ACC TCA TAG
TAT ACC
CTA C W ACT TTA TGT CAT TAT
AM
TE AGA AAT CCA
TSr CGA ClC
ATT
CTT A X GCC
3540
GAC T M TCT A X TAA CGT
3600
AAC CAT CCT CAA RIT W CAC TTA CCT GAT CTT TAT
V I ? AAC GTG GTA CC
GM
MA
GM;
T T G GTT CM
3660
3600
FIG. 4"continued
Primary Structure
1278
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 theINOl 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 andMethods,” and the results are displayed in Table IV. The amino terminus of the predicted protein (Fig. 4) (Met-Thr-Glu-Asp-Asn-Ile-AlaPro) when compared to the chemically determined amino terminus of MI-1-P synthase (TableIV) (Met-(Ser/Thr)-GluAsp-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 databasefor homologies to theINOl coding sequence. No significant homologies to any reported protein sequences
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. A n Analysis of the DNA Sequence of theOpen 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 ithas featuresthat could function in theinsertion 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 Analysisof Strains Bearing Gene Disruptions 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 andMethods” and illustrated inFig. 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 Peptides I NO 1 gene 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 1Kb -c molecular level (Fig. 8). The inositol-requiring (Ino-) spores, FIG. 5. Computer analysis of the DNA fragment containing the INOl gene shows that there are three potential peptidesderived from sporulation of the diploid bearing the INOl which couldbe read in the same reading frame as the largest disruption, were subjected to genetic complementation analyopen reading frame, corresponding to the INOl gene. sis. Haploid strains of genotype inol-13 (Table I) (inol-13 is
-
U
TABLE I1 Codon usage and amino acid composition predictedfrom the DNA sequence of INOI, the structural gene for MI-I-P synthase Codon usaee AAA ( L y s ) :
21
ACA(Thr): 5 AGA(Arg): 8 ATA(I1e): 2 C A A ( G 1 n ) : 17 CCA(Pro): 5 CGA(Arg): 4 CTA ( L e u ) : 2 G A A ( G 1 u ) : 17 GCA ( A l a ) : 9 GGA(G1y): 3 GTA(Va1): 7 TAA(ST0P): 1 TCA(Ser): 3 TGA(ST0P): 0 T T A ( L e u ) : 14
AAC(Asn): ACC(Thr): AGC(Ser): ATC(I1e): CAC(His): CCC(Pro): CGC ( A r g ) : CTC ( L e u ) : GAC(Asp): GCC ( A l a ) : GGC ( G l y ) : GTC(Va1): TAC(Tyr): TCC(Ser): TGC(Cys): TTC ( P h e ) :
14 10 4 16 4 5 4 6 15 6 9 8 12 13 4
10
AAG(Lys): ACG(Thr): AGG ( A r g ) : ATG(Met): CAG(G1n): CCG(Pro): CGG ( A r g ) : CTG(Leu): GAG(G1u): GCG(A1a): GGG ( G l y ) : GTG(Va1): TAG(ST0P): TCG ( S e r ) : TGG ( T r p ) : TTG(Leu):
23 5 5 11 7 4 2 10 14 4 2 7
0 1 9 15
AAT(Asn): ACT(Thr): AGT(Ser): ATT(I1e): CAT ( H i s ) : CCT(Pro): CGT ( A r g ) : C T T( L e u ) : GAT(Asp): GCT(A1a): GGT(G1y): GTT(Va1): TAT(Tyr): TCT(Ser): TGT(Cys): TTT(Phe):
21 10 6 17 6 8 0 9 19 13 11 19 10 15 2 11
Predicted amino acid comDosition
A l a : 32 (5.8%) Cys: 6 ( 1 . 1 % ) H i s : 10 ( 1 . 8 % ) Met: 11 ( 2 . 0 % ) T h r : 30 ( 5 . 4 % )
23 24 35 21 Trp: 9
Arg: Gln: Ile: Phe:
(4.2%) (4.3%) (6.3%) (3.8%) (1.6%)
A s n : 35 ( 6 . 3 % ) G l u : 31 ( 5 . 6 % ) L e u : 56 ( 1 0 . 1 % ) Pro: 22 ( 4 . 0 % ) T y r : 22 ( 4 . 0 % )
Asp: Gly: Lys: Ser: Val:
34 25 44 42 41
(6.1%) (4.5%)
(8.0%) (7.6%) (7.4%)
Primary Structure
of Yeast myo-Inositol-1
-phosphate Synthase
1279
a missense allele derived from mutagenesis of a wild-type strain with ethylmethane sulfonate (10)) were crossed to Inostrains carrying the INOl gene disruption. Diploid strains +2 produced from such crosses were inositol auxotrophs. Thus, the INOl gene disruption failed to complement existing in01 +I alleles, providing additional genetic confirmation of the identity of the large open reading frame as theINOl locus. +O Cellular extracts made from haploid strains carrying the -1 INOl gene disruption were subjected to Western blot analysis (39). Such strains failed to express any material cross-reactive -2 to MI-1-P synthase antibody (Fig. 9). In comparison, the control strains, both wild-type and a haploid strain bearing -37 - ........................................................... the inol-13 allele (inol-13 strains produce inactive MI-1-P Hydrophobic -4 , I , I , I I I I I I I I , I , I ( I synthase) did express material cross-reactive to anti-MI-1-P 0.0 0.1 0.2 0 3 0.4 0.5 0 6 0.7 0.8 0 9 1.0 synthase. As expected, the regulatory mutant ino4, which fails Fraction of Length to derepress the enzyme subunit (9),does not express material FIG. 6. Hydrophobicity plot of the INOl gene product as cross-reactive to MI-1-Psynthase. determined by the DNA Inspector I1 program. 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 disrupTABLEI11 tion. Haploid strains carrying the disruption of the small open Comparison of the chemically determined amino acid composition of MI-1-P synthase purified from wild-type yeast with the predicted reading frame were found to be viable. They were not auxotrophic for inositol and had no evident growth defect. Northamino acid composition generated from computer analysisof the ern blot analysis (41) confirmed the absence of the transcript DNA seauence of the INOl gene of the small open reading frame (Fig. 10) in haploid cells Composition Amino acid of Composition average predicted from DNA carrying the disruption. Strainscarrying the disruption of the MI-l-P svnthase chemically determined sequence analysis small open reading frame were shown to excrete inositol by a % total nmol bioassay (14). The excretion phenotype was foundto be 1.8 Histidine 1.8 genetically cis-dominant when diploids heterozygous for the 7.8 Lysine 8.0 disruption were constructed. However, Western blot analysis 3.0 Arginine 4.2 (Fig. 11) of MI-1-P synthase expression in haploid strains *12.7 Aspartic acid 6.1 carrying the disruption of the small open reading frame reThreonine 5.9 5.4 vealed that MI-1-P synthase expression was repressed nor6.8 Serine 7.6 *10.8 5.6 Glutamic acid mally in cells grown in the presence of inositol. Thus, disrup5.4 Proline 4.0 tion of the small open reading frame led to excretion of 6.8 Glycine 4.5 inositol but did not appear to affect regulation of MI-1-P 7.5 Alanine 5.8 synthase. 0.9 1.1 Half-cystine The phospholipid composition of both categories of gene Valine 7.6 7.4 disruption mutants was analyzed. No significant differences Methionine 1.6 2.0 5.7 Isoleucine 6.3 in thephospholipid compositions of the gene disruptants were Leucine 10.1 8.7 found when compared to thewild-type composition (data not Tyrosine 4.0 3.7 shown). Hydrophilic
...........................................................
Phenylalanine Tryptophan Asparagine Glutamine
4.0
3.8
* Value contained in
6.3
aspartic acid * Value contained in glutamic acid
4.3
1.6
TABLEIV Primary sequence of the amino terminus ofMI-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
1
HPLC I1
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.
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 synthasewith that determined by molecular analysis. This comparison shows excellent agreement of the two compositions providing direct substantiationthat 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 atposition 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
Primary S t r u c t u r e of Yeast myo-Inositol-1-phosphate Synthase
1280
TABLEV Conserved sequences in the flanking regions of the INOl gene 5' Promoter region Distance from ATG
INOl equivalent
Consensus sequence GGPyCAAG CAAT TATA
PumTGPuXT
GGCCAAG
-274
CAAT TATAAATT TATTTAAT ACAAAACA CTAAATT
-140 -122 -247 -3 -70
Reference
Function
Modulator Yeast promoter mRNAselectionsite
43-45
Translationalstartsite
47
43 4 3 , 1 5 . 46
3' Termination region
Consensus sequence f o r
eukaryoticgenes (48)
INOl e q u i v a l e n t
TAA TAG TGA
140basepairs (T-rich)
TAA
60basepairs (T-rich)
(AT-rich)
TAGT TATGT (AT-rich)
TAG
TAGT
TIT
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-terminalsequence
Small o p e n r e a d i n g frame
Thr-Leu-Leu-Asn-Leu-Leu-Leu-Phe-Leu-Leu Leu-Phe-Phe-Pro-Ala-Ile-Ile
Pancreatic secretoryproteins (49)
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
GeneEproteinof OX-174 (50)
B n L F l p r o t e i n o f E p s t e i n - B a r r v i r u s (51) Gag p o l y p r o t e i n o f t h e f e l i n e s a r c o m a v i r u s ( 5 2 )
+4
-
+3-
-
+2
Hydrophilic
...........................................................
4
I 1
2
3
4
5
6
FIG.8. Southern blot analysis (38)was used to locate the
J
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 insertionof the LEU2 gene into the Hydrophobic - 4 1 , I , , I , I , I , I I I ' I ' INOl region (as illustrated in Fig. 3) generated a fragment of approx0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 imately 7.9 kb (upper arrow). To demonstrate the genetic linkage of Fraction of Length the leucine prototrophy to the INOI locus, a transformed diploid was sporulated and 20 tetrads were analyzed. The leucine prototrophy in FIG.7. Hydropkylicity plot of the largest open reading frame upstream of the INOZ gene shows thatit has features the meiotic products is genetically linked to the I N 0 1 locus and cosegregates withthe larger restriction fragment producedby the gene of a membrane-associated protein. disruption. That is, the Ino- phenotype co-segregated with the Leu+ phenotype in all tetrads. Lanes I and 6 contain DNA isolated from 1380 is also consistent with the findingby Hirsch (57) that the disrupted diploid. These strains possess both restriction fragthe major INOl transcript starts five nucleotides upstream ments. Lanes 2 and 3 contain DNA from spores that are phenotypiwhile lanes 4 and 5 contain cally Leu- (smaller restriction fragment), from the start of translation (i.e. a t position 1375 relative to DNA isolated from Leu' spores (larger restriction fragment produced Fig. 4). Codon usage analysis was consistent with the identi- by gene disruption). fication of a moderately expressed yeast protein (41,42). The hydrophilicity analysis of MI-1-P synthase illustrated 1-Psynthase sequence suggests that the protein isprobably in Fig. 6 has confirmed that MI-1-P synthase, as expected for not associated with membrane. The determination of the a cytoplasmic enzyme, has no extensiveregions of hydropho- primary sequence of MI-1-P synthase, however, opens the bicity. Initially, the enzyme was purified using its affinity to possibility of more detailed studies in the future of the struchydrophobic resins (9), suggesting that itmight be unusually ture of the enzyme and may permit localization of active sites hydrophobic for a cytoplasmic enzyme. However, the absence within the primarysequence. This enzyme is known to cataof extensive stretchesof hydrophobic amino acids in theMI- lyze a complex series of reactions that involve a t least three -3-
,
Primary Structure of Yeast myo-Inositol-1 -phosphate Synthase
1281
c
1
2
3
4
5
FIG. 11. Analysis of regulation of MI-1-P synthase in cells bearing thedisruption of the small open reading frame. West-
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-I3strain 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 crossreactive 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 p M 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 synthasesubunit.
ern blot analysis wasused 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 p M 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 p M 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 thestart 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 presentin most eukaryotic genes and is implicated in theeukaryotic RNA polymerase recognition site (43). Anothersequence 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 I 2 3 4 to thestart of translation (i.e. nucleotide 1240 in Fig. 4). The 133-amino acid open reading frame adjacent to INOl FIG. 10. Northern blot analysis of strains bearing thedisruption of the small open reading frame. RNA was extracted has been shown to encode a mRNA species of0.6 kb (16). from cells grownfrom the four spore colonies of a single tetrad The region 5' to the first ATG of this open reading frame is dissected from the diploid strain bearing the gene disruption. Cells filled with TATA-like sequences. For example, TATAATT is were grownin the absence of inositol. Lane 1, spore colony A, carrying located at position 529 (Fig.4), TATATAAA a t position 431, the disruption (Leu+).Lane 2, spore colony B (Leu-) no disruption. Lane 3, spore colony C (Leu-) no disruption. h n e 4, spore colony D and TATATA at position 250. A potential poly(A) addition carrying the disruption (Leu'). Equal amounts of total RNA were site (AAATAA) is located a t position 431. Also, the sequence applied to each lane. The probe used spanned the entire INOl region AGCCAGCTGCAG that is located at position 465 has been (Fig. 2). Note the presence of the INOl transcript (upper arrow) in identified from mammalian sources as a phorbol ester-binding all four lanes. The smaller transcript (lower arrow) is missing in the site (53). Further analysis will be required before the funcstrains A and D carrying the disruption (lanes 1 and 4). tion(s) of these sequences can be determined. However, the putative peptide encoded by this open reading frame has an partial reactions (5-7). It is not known how many active sites intriguing structure. The hydrophilicity plot shown in Fig. 7 there arewithin MI-1-P synthase nor how they arepositioned indicates that there are long stretches of hydrophobic residues relative to one another. Future analysis of the positioning of that could be readily associated with a membrane. Although the in01 mutations that destroy catalytic activity of the en- a rigorous determination of the transmembrane disposition of zyme relative to the primary sequence may permitthese a proteingenerally requires the use of macromolecular probes such as antibodies, proteases, and nonpenetrating or selective questions to be addressed. Comparison of the primary sequence of the INOl gene labeling reagents, the availability of complete primary seproduct MI-1-P synthase toproteins found in Bionet's data- quences for numerous membrane proteins has allowed tentabank revealed no homology to any otherpreviously analyzed tive predictions of their transmembrane disposition to be protein. It will be interesting in the future to compare the made on the basis of hydropathy plots (54). Search of the structure of the enzyme from yeast to other proteins which NBRF protein database for proteins homologous to the 133utilize glucose 6-phosphate as substrate or NADH as cofactor. 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 2. Eisenberg, F., Jr., and Bolden, A. H. (1962) Biochem. Biophys. Res. Commun. 1 2 , 72-77 data bank (Table VI). In fact, the hydrophobic stretch of 3. Loewus, F. A., and Kelly, S. (1962) Biochem. Biophys. Res. Comamino acid residues starting at position 605 following the mun. 7,204-208 putative signal sequence is quite homologous to a sequence in 4. Eisenberg, F., Jr., Bolden, A. H., and Loewus, F. A. (1964) the amino terminus of rat liver cytochrome P-450 PB-4 (55). Biochem. Bwphys. Res. Commun. 14,419-424 The sequence (Met-Glu-Pro-(Ser)-Ile-Leu-Leu-Leu-Leu-Ala5. Kiely, D. E., and Sherman, W. R. (1975) J. Am. Chem. SOC. 97, 6810-6814 Leu-Leu-Val-Gly-Phe-Leu-Leu-Leu-Leu-Val) of rat liver cy6. Maeda, T., and Eisenberg, F., Jr. (1980) J. Biol.Chem. 2 5 5 , tochrome P-450 PB-4 has been shown to anchor the protein 8458-8464 to the endosplasmic reticulum (55). The hydrophobic region 7. Loewus, F. A., and Loewus,M.W. (1983) Annu. Reu. Plant in the amino-terminal region of the 133-amino acid open Physiol. 34,137-161 reading frame also appears to share homologies to similar 8. Sherman, W. R.,Stewart, M.A., and Zinbo, M. (1969) J. Bwl. regions found in the hypothetical BNLFl protein of EpsteinChem. 244,5703-5708 Barr virus, the gag polyprotein of the feline sarcoma virus, 9. Donahue, T. F., and Henry, S. A. (1981) J. Bwl.Chem. 2 5 6 , 7077-7085 the gene E protein of 9x174, and theamino-terminal sequence 10. Culbertson, M. R.,and Henry, S. A. (1975) Genetics 80,23-40 of precursors to pancreatic secretory proteins (Table VI). ", S. A. (1976) J. Gene disruptions designed to eliminate, individually, 11. Culbertson, M.R., Donahue. T. F.. and Henrv. Bacterwl.' 126,232-250 . expression of the open reading frame upstream of the ZNOl 12. Klig, L. S., and Henry, S. A. (1984) Proc. Natl. Acad. Sci. U.S. A. gene and the IN01 gene itself proved to be viable. Unlike 81,3816-3820 most yeast loci, but similar to theyeast HIS1 locus, the IN01 13. Greenberg, M. L., Goldwasser, P., and Henry, S. (1982) Mol. Gen. Genet. 1 8 6 , 157-163 locus lacks allelic representatives that are suppressible by known nonsense suppressors (56). This finding suggested that 14. Greenberg, M. L., Reiner, B., and Henry, S. A. (1982) Genetics 100,19-33 premature termination of translation of the IN01 gene prodB. S., and Henry, S. A. (1984) Mol. Cell. Bwl. 4 , 2479uct MI-1-P synthase might be incompatible with cell viability. 15. Loewy, 2485 Testing of this hypothesis was a major motivation for creating 16. Hirsch, J. P., and Henry, S. A. (1986) Mol. Cell. Biol. 6 , 3320null mutations at ZNOl. However, genetic complementation 3328 analysis as well as Western blotanalysis confirmed the exist- 17. Homann, M. J., Henry, S. A., and Carman, G.M. (1985) J. Bacteriol. 163,1265-1266 ence of viable null mutations at INOl. The null mutants are simply inositol auxotrophs similar in growth and phospholipid 18. Klig, L. S., Homann, M. J., Carman, G.M., and Henry, S. A. (1985) J. Bacteriol. 162, 1135-1141 composition to other inol alleles. Thus, failure to detect 19. Carson, M.A., Atkinson, K. D., and Waechter, C. J. (1982) J. nonsense mutants at theIN01 locus remains an unexplained Biol. Chem. 257,8115-8121 phenomenon. 20. Carson, M.A., Emala, M., Hogsten, P., and Waechter, C. J. The disruption of the small open reading frame upstream (1984) J. Biol. Chem. 259,6267-6273 of the ZNOl gene is also viable and causes the cell to excrete 21. Carter, J. R., Jr. (1968) J. Lipid Res. 9 , 748-754 inositol, a phenotype which is associated with mutants defec- 22. Bailis, A. M., Carman, M. A., and Henry, S. A. (1986) Mol. Cell. Biol. 7,167-176 tive in regulation of phospholipid biosynthesis (13, 14). The 23. Steiner, S., and Lester, R. L. (1972) Biochim. Biophys. Acta2 6 0 , excretion phenotype is genetically cis-dominant in the strain 82-87 disrupted in the small open reading frame, whereas most 24. Steiner, M. R., and Lester, R. L. (1972) Biochim. Biophys. Acta other Opi- mutantsare recessive.However, unlike other 260,222-243 regulatory mutants with the inositol excretion phenotype such 25. Steiner, S., and Lester, R.L. (1972) J. Bacteriol. 1 0 9 , 81-88 as opil (13), synthesisof MI-1-P synthase isrepressed in the 26. Waechter, C. J., and Lester, R. L. (1971) J. Bacteriol. 105,837410 presence of inositol in cells carrying the gene disruption. The insertion of the LEU2 gene in the region adjacent to theZNOl 27. Waechter, C. J., and Lester, R. L. (1973) Arch. Biochem. Biophys. 158,401-410 gene may have affected INOl transcription, leading to over- 28. Kennedy, E. P., and Weiss, S. B. (1956) J. Bwl. Chem. 222,193production of inositol. At present, we believe that this is a 214 probable explanation for the inositol excretion phenotype 29. Barnett, J. E. G., Brice, R. E., and Corina, D. L. (1970) Biochem. J. 119,183-186 observed in strains bearing the disruption of the small open reading frame. Because the disruption of the small open 30. Steinman, H. M. (1978) J . Biol. Chem. 253,8708-8720 reading frame had no evident effect upon growth, it may be 31. Laemmli, U.K. (1970) Nature 227,680-685 M. W., Lujan, E., Ostrander, F., and Hood, W. E. concluded that it does not encode aprotein essential for 32. Hunkapiller, (1983) Methods Enzymol. 9 1 , 227-237 growth under the laboratory conditions described inthis 33. Henikoff, S. (1984) Gene (Amst.) 28,351-359 report. No further conclusion can be drawn at thistime about 34. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U.S. A. 74,5463-5467 the function of its gene product in phospholipid biosynthesis or regulation or in other cellular processes. Future experi- 35. Sanger, F., and Coulson, A. R. (1978) FEBS Lett. 8 7 , 107-110 ments designed to identify the gene product (gene fusions, 36. Vieira, J., and Messing, J. (1982) Gene (Amst.) 1 9 , 259-268 Rothstein, R. J. (1983) Methods Enzynwl. 1 0 1 , 202-211 antibody production) would give a better understanding of 37. 38. Southern, E. M. (1975) J. Mol. Biol. 98,503 the role of the product of this gene in yeast cell biology. 39. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. .
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. REFERENCES 1. Majerus, P. W., Connolly, T. M., Deckmyn, H., Ross, T. S., Bross, T. E., Ishii, H., Bansal, V. S., and Wilson, D. B. (1986) Science 234,1519-1526
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52. Laprevotte, I., Hampe, A., Sherr, C., and Galibert, F. (1984) J. Virol. 50, 884-894 53. Comb, M., Birnberg, N., Seasholtz, A., Herbert, E., and Goodman, H. (1986) Nature 323,353-356 54. Hopp, T. P., and Woods, K. R. (1981) Proc. Natl. Acad. Sci. U.S. A. 78,3824-3828 55. Chiarandini-DeLemos, C., Frey, A. B., Sabatini, D. D., and Kreibach, G. (1987) J. Cell. Biol. 104, 209-219 56. Donahue, T. F., and Henry, S. A. (1981) Genetics 98,491-503 57. Hirsch, J. (1987)cis and trans Acting Regulationof the IN01 Gene of Saccharomycescereuisiae. Ph.D. thesis, Albert Einstein College of Medicine, Bronx, NY
