The mechanism of interallelic complementation at the INO1 locus in yeast: Immunological analysis of mutants

Majumder, Arun Lahiri; Duttagupta, Swadesh; Goldwasser, Paul; Donahue, Thomas F.; Henry, Susan A.

doi:10.1007/bf00352503

Cited by 20 publications

(14 citation statements)

References 13 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Cells containing recombinant plasmids, pGEMT-Easy and pET-20b(ϩ), were supplemented with 100 g/ml ampicillin. Yeast strains were routinely grown at 30°C in YEPD and complete synthetic media as required (5).…”

Section: Methodsmentioning

confidence: 99%

“…This mechanism is followed by all myo-inositolproducing organisms throughout the phylogenetic lines, and MIPS has been identified as an evolutionarily conserved protein (4). The structural gene coding for cytosolic MIPS, termed INO1, was first identified in yeast, Saccharomyces cerevisiae (5,6) and cloned by Klig and Henry (7). Subsequently, the complete nucleotide sequence of the full-length INO1 gene from S. cerevisiae was reported by Johnson and Henry (8).…”

mentioning

confidence: 99%

See 1 more Smart Citation

A Novel Salt-tolerant l-myo-Inositol-1-phosphate Synthase from Porteresia coarctata (Roxb.) Tateoka, a Halophytic Wild Rice

Majee

Maitra

Dastidar

et al. 2004

Journal of Biological Chemistry

Self Cite

173

View full text Add to dashboard Cite

L-myo-Inositol-1-phosphate synthase (EC 5.5.1.4, MIPS), an evolutionarily conserved enzyme protein, catalyzes the synthesis of inositol, which is implicated in a number of metabolic reactions in the biological kingdom. Here we report on the isolation of the gene (PINO1) for a novel salt-tolerant MIPS from the wild halophytic rice, Porteresia coarctata (Roxb.) Tateoka. Identity of the PINO1 gene was confirmed by functional complementation in a yeast inositol auxotrophic strain. Comparison of the nucleotide and deduced amino acid sequences of PINO1 with that of the homologous gene from Oryza sativa L. (RINO1) revealed distinct differences in a stretch of 37 amino acids, between amino acids 174 and 210. Purified bacterially expressed PINO1 protein demonstrated a salt-tolerant character in vitro compared with the salt-sensitive RINO1 protein as with those purified from the native source or an expressed salt-sensitive mutant PINO1 protein wherein amino acids 174 -210 have been deleted. Analysis of the salt effect on oligomerization and tryptophan fluorescence of the RINO1 and PINO1 proteins revealed that the structure of PINO1 protein is stable toward salt environment. Furthermore, introgression of PINO1 rendered transgenic tobacco plants capable of growth in 200 -300 mM NaCl with retention of ϳ40 -80% of the photosynthetic competence with concomitant increased inositol production compared with unstressed control. MIPS protein isolated from PINO1 transgenics showed salt-tolerant property in vitro confirming functional expression in planta of the PINO1 gene. To our knowledge, this is the first report of a salt-tolerant MIPS from any source.Inositols are six-carbon cyclohexane hexitols found ubiquitously in the biological kingdom, and its metabolism plays a vital role in growth regulation, membrane biogenesis, osmotolerance, and in many other processes. As phosphorylated derivatives, its role as a phosphorus store and as a "second messenger" in signal transduction pathways has long been recognized. myo-Inositol, physiologically the most favored stereoisomer among the eight possible geometric isomers of inositol, also enters into an array of biochemical reactions having diverse functions in cellular metabolism both as free and conjugated and phosphorylated or methylated forms (1-3).The primary enzyme for the synthesis of L-myo-inositol 1-phosphate from glucose 6-phosphate is L-myo-inositol-1-phosphate synthase (EC 5.5.1.4; referred to as MIPS), 1 which synthesizes L-myo-inositol 1-phosphate through an internal oxidoreduction reaction involving NAD ϩ . Free inositol is generated by dephosphorylation of the MIPS product by a specific Mg 2ϩ -dependent inositol-1-phosphate phosphatase (EC 3.1.3.25). This mechanism is followed by all myo-inositolproducing organisms throughout the phylogenetic lines, and MIPS has been identified as an evolutionarily conserved protein (4). The structural gene coding for cytosolic MIPS, termed INO1, was first identified in yeast, Saccharomyces cerevisiae (5, 6) and cloned by Klig and Henry (7)....

show abstract

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

A Novel Salt-tolerant l-myo-Inositol-1-phosphate Synthase from Porteresia coarctata (Roxb.) Tateoka, a Halophytic Wild Rice

Majee

Maitra

Dastidar

et al. 2004

Journal of Biological Chemistry

Self Cite

173

View full text Add to dashboard Cite

show abstract

“…The genetics of inositol biosynthesis and its regulation have been elaborately studied in S. cerevisiae Nikoloff and Henry, 1991;White et al, 1991). The structural gene for the cytosolic I-1-P synthase was identified as Inol for the first time in S. cerevisiae Majumder et al, 1981). Subsequently, the Inol gene was cloned and sequenced from a number of fungal and plant sources Johnson and Henry, 1989;Smart and Fleming, 1993;Abu-Abied andPlant Physiol.…”

mentioning

confidence: 99%

L-myo-lnositol 1-Phosphate Synthase from Plant Sources (Characteristics of the Chloroplastic and Cytosolic Enzymes)

et al. 1997

View full text Add to dashboard Cite

i-myo-inositol 1 -phosphate synthase (EC 5.5.1.4) from cyanobacteria1 (Spirulina platensis), alga1 (Euglena gracilis), and higher plant (Oryza sativa, Vigna radiata) sources was purified to electrophoretic homogeneity, biochemically characterized, and compared. Both chloroplastic and cytosolic forms of the enzyme were detected in E. gracilis, O. sativa, and V. radiata, whereas only the cytosolic form was detected in' streptomycin-bleached or chloroplastic mutants of E. gracilis and in S. platensis. Both the chloroplastic and cytosolic forms from different sources could be purified following the same three-step chromatographic protocol. i-myo-inositol 1 -phosphate synthases purified from these different sources do not differ significantly with respect to biochemical and kinetic parameters except for the molecular mass of the chloroplastic and cytosolic native holoenzymes, which appear to be homotetrameric and homotrimeric associations of their constituent subunits, respectively. Monovalent and divalent cations, sugar alcohols, and sugar phosphates are inhibitory to the enzyme activity. N-ethylmaleimide inhibition of synthase activity could be protected by the combined presence of the substrate glucose-6-phosphate and cofactor. NAD+. Antibody raised against the cytosolic enzyme from E. gracilis immunoprecipitates and cross-reacts with both chloroplastic and cytosolic forms from the other sources studied.Inositols are 6-C cyclohexane hexitols found ubiquitously in biological systems. Of the eight possible geometrica1 isomers, myo-inositol is the most abundant and occupies a central position in carbohydrate metabolism, being the precursor of a number of metabolic products such as inositol phosphates, phosphoinositides, cell wall polysaccharides, a number of pethylated derivatives, and IAA conjugates. As a free cyclitol, myo-inositol has been identified as a compound required for normal growth and development of fungal and plant tissue culture cells (Loewus and Dickinson, 1982;Loewus and Loewus, 1983;Biswas et al., 1984;Loewus, 1990). Depletion of the normal cellular level of inositol has been shown to lead to a loss of cell viability in the yeast Saccha-

show abstract

“…The inositol-requiring strain LT13 (inol-13 leu2-) was transformed with the clone bank of yeast DNA on vector YEp13. The inol allele (inol-13) present in strain LT13 was selected for these experiments because it is a stable missense mutation (1,8). Transformants were selected sequentially, using leucine and then inositol prototrophy to avoid the possibility of "inositol-less death" (7) prior to the plasmid expressing the gene(s) it contained.…”

Section: Resultsmentioning

confidence: 99%

“…The tetrad dissection was carried out on leucine-deficient agar and, thus, all surviving spores received the plasmid. The genotype of each spore relative to the inol or ino4 alleles segregating in the cross was determined by allele testing (7,8) derivative colonies that had lost the plasmid. Colonies that had lost the plasmid were isolated by permitting subcultures of each spore to grow under conditions that are nonselective for the plasmid (i.e., in the presence of leucine and inositol).…”

mentioning

confidence: 99%

Isolation of the yeast INO1 gene: located on an autonomously replicating plasmid, the gene is fully regulated.

Klig¹,

Henry²

1984

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The Saccharomyces cerevisiae gene, INO), encoding the highly regulated enzyme, myo-inositol-l-phosphate synthase [1L-myo-inositol-l-phosphate Iyase (isomerizing), EC 5.5.1.4], was isolated by genetic complementation. The cloned sequence was shown to complement two independent INO) alleles (inol-5 and inol-13). One of these mutants (inol-5) fails to make any material that is crossreactive with antibody to the wild-type inositol-l-phosphate synthase. The cloned DNA restored not only inositol prototrophy to this mutant but also its ability to make material crossreactive with anti-inositol-l-phosphate synthase antibody. The sequence on an integrative plasmid was also shown to recombine with the INO) locus, thereby confirming its genetic identity. The DNA was subcloned and used for Southern blot analysis, revealing that the cloned DNA (5.4 kilobases long) represents a unique sequence in the yeast genome. Inositol-l-phosphate synthase was fully regulated when its gene was located extrachromosomally on the autonomously replicating plasmid. In cells (inol ) containing the cloned INOl gene on a high-copy-number plasmid, the enzyme was fully repressible. Furthermore, the gene product was not expressed when the plasmid was transferred into a strain containing an ino4 mutation, which also prevents expression of chromosomal copies of INOJ.These results establish that the intact structural gene and associated regulatory components have been isolated and that positioning of the gene in its normal chromosomal location is not required for full regulation of inositol-l-phosphate synthase.The synthesis of inositol, a critical phospholipid precursor, is a highly regulated process in yeast. myo-Inositol-1-phosphate synthase [Ins1P synthase; 1L-myo-inositol-1-phosphate lyase (isomerizing), EC 5.5.1.4], a repressible cytoplasmic enzyme (1), is the major biosynthetic enzyme in this pathway. This enzyme has been purified from several eukaryotic organisms, including mammals (2) and yeast (1). In yeast (1), the enzyme has a molecular size in excess of 200,000 daltons and is a multimer of identical subunits of 62,000 daltons. The reaction this enzyme catalyzes results in the conversion of glucose 6-phosphate to inositol 1-phosphate. The reaction includes a ring closure and a coupled oxidation and reduction in which NAD is a cofactor (1). Despite intensive biochemical analysis, the precise reaction mechanism remains to be proven, although it has been proposed to consist of three partial reactions involving the intermediates 5-ketoglucose 6-phosphate and inosose-2 1-phosphate (3).In yeast, InslP synthase is the gene product of the INOI gene (1), but a large number of other genes are known to affect its expression. For example, the ino2 and ino4 mutations, which are unlinked to INOI, prevent expression of InslP synthase, while the opil, opi2, and opi4 mutations render the enzyme constitutive (4). Several lines of evidence suggest that some of these unlinked regulatory genes are involved in the coordination of InslP synthase expression...

show abstract

The mechanism of interallelic complementation at the INO1 locus in yeast: Immunological analysis of mutants

Cited by 20 publications

References 13 publications

A Novel Salt-tolerant l-myo-Inositol-1-phosphate Synthase from Porteresia coarctata (Roxb.) Tateoka, a Halophytic Wild Rice

A Novel Salt-tolerant l-myo-Inositol-1-phosphate Synthase from Porteresia coarctata (Roxb.) Tateoka, a Halophytic Wild Rice

L-myo-lnositol 1-Phosphate Synthase from Plant Sources (Characteristics of the Chloroplastic and Cytosolic Enzymes)

Isolation of the yeast INO1 gene: located on an autonomously replicating plasmid, the gene is fully regulated.

Contact Info

Product

Resources

About