SUC2 Gene Research Articles

Organization of the SUC gene family in Saccharomyces.

The SUC gene family of yeast (Saccharomyces) includes six structural genes for invertase (SUC1 through SUC5 and SUC7) found at unlinked chromosomal loci. A given yeast strain does not usually carry SUC+ alleles at all six loci; the natural negative alleles are called suc0 alleles. Cloned SUC2 DNA probes were used to investigate the physical structure of the SUC gene family in laboratory strains, commercial wine strains, and different Saccharomyces species. The active SUC+ genes are homologous. The suc0 allele at the SUC2 locus (suc2(0) in some strains is a silent gene or pseudogene. Other SUC loci carrying suc0 alleles appear to lack SUC DNA sequences. These findings imply that SUC genes have transposed to different chromosomal locations in closely related Saccharomyces strains.

Nucleotide sequence of the yeast SUC2 gene for invertase.

The yeast SUC2 gene is a structural gene for both the secreted and intracellular forms of invertase. We have determined the nucleotide sequence of the coding region and the 5' and 3' flanking regions. The coding regions for the signal peptide-containing precursor to secreted invertase and for the intracellular invertase begin at different initiation codons within the SUC2 gene but share the same reading frame. The amino acid sequences predicted for the two forms of invertase from the nucleotide sequence are consistent with the properties of the purified enzymes. Potential sites for glycosylation of the secreted invertase are identified.

Two differentially regulated mRNAs with different 5′ ends encode secreted and intracellular forms of yeast invertase

The SUC2 gene of yeast (Saccharomyces) encodes two forms of invertase: a secreted, glycosylated form, the synthesis of which is regulated by glucose repression, and an intracellular, nonglycosylated enzyme that is produced constitutively. The SUC2 gene has been cloned and shown to encode two RNAs (1.8 and 1.9 kb) that differ at their 5′ ends. The stable level of the larger RNA is regulated by glucose; the level of the smaller RNA is not. A correspondence between the presence of the 1.9 kb RNA and the secreted invertase, and between the 1.8 kb RNA and the intracellular invertase, was observed in glucose-repressed and -derepressed wild-type cells. In addition, cells carrying a mutation at the SNF1 locus fail to derepress synthesis of the secreted invertase and also fail to produce stable 1.9 kb RNA during growth in low glucose. Glucose regulation of invertase synthesis thus is exerted, at least in part, at the RNA level. A naturally silent allele ( suc2°) of the SUC2 locus that does not direct the synthesis of active invertase was found to produce both the 1.8 and 1.9 kb RNAs under normal regulation by glucose. A model is proposed to account for the synthesis and regulation of the two forms of invertase: the larger, regulated mRNA contains the initiation codon for the signal sequence required for synthesis of the secreted, glycosylated form of invertase; the smaller, constitutively transcribed mRNA begins within the coding region of the signal sequence, resulting in synthesis of the intracellular enzyme.

Distinct repressible mRNAs for cytoplasmic and secreted yeast invertase are encoded by a single gene

We have studied regulation of invertase putative structural genes ( SUC) in S. cerevisiae and the synthetic relationship between secreted, glycosylated invertase (E.C.3.2.1.26) and the cytoplasmic, nonglycosylated form of the enzyme. Using immunoprecipitation and gel electrophoresis, we have analyzed invertase polypeptides and glycopeptides synthesized in vitro and in vivo. Analysis of size-fractionated mRNA from a SUC2 strain has shown that three mature, catabolite-repressible mRNA species direct the in vitro synthesis of three invertase polypeptides that have differing molecular weights. Two of these polypeptides, P63 and P62 (63 and 62 kd), are larger than the polypeptide of the secreted enzyme and are cotranslationally processed by microsomal membranes in vitro to yield secreted invertase glycopeptides (GP90 and GP87). The smallest polypeptide, P60 (60 kd), which comigrates electrophoretically with cytoplasmic invertase, is not processed. Posttranslationally, a microsomal-membrane detergent extract removes approximately 20 amino acids from P62 but not from P60. In vitro translations of mRNAs from a genetically confirmed suc3 mutant strain, from the parental SUC3 strain and from derivative meiotic segregants have shown that the three polypeptides (and therefore three mRNA species) are encoded by one gene. Analysis of in vivo radiolabeled invertase from the same SUC3 and suc3 strains has verified that the SUC3 locus contains the structural gene for secreted and cytoplasmic invertase. Through the derepressed synthesis of multiple primary or processed transcripts, the SUC2 and SUC3 genes are regulated to produce multiple invertase polypeptides. The larger two polypeptides appear to be processed and secreted to yield glycosylated invertase, while the smallest remains in the cytoplasm.

SUC1 gene of Saccharomyces: a structural gene for the large (glycoprotein) and small (carbohydrate-free) forms of invertase.

Saccharomyces cerevisiae revertant strain D10-ER1 has been shown to contain thermosensitive forms of the large (glycoprotein) and small (carbohydrate-free) invertases and a very low level of the small enzyme, along with a wild-type level of the large form (T. Mizunaga et al., Mol. Cell. Biol. 1:460-468, 1981). These characteristics cosegregated in crosses of the revertant strain with wild-type sucrose-fermenting (SUC1) or nonfermenting (suc0) strains. In addition, there is tight linkage between sucrose and maltose fermentation in revertant D10-ER1 (characteristic of the SUC1 and MAL1 genes). From this we infer that a single reversion event is responsible for the several changes observed in D10-ER1, and that this mutation maps within or very close to the SUC1 gene present in the ancestor strain 4059-358D. The revertant SUC1 allele in D10-ER1 (termed SUC1-R1) was expressed independently of the wild-type SUC1 gene when both were present in diploid cells. Diploids carrying only the wild-type or the mutant genes synthesized invertases with the characteristics of the parental Suc+ haploids. The possibility that a modifier gene was responsible for the alterations in the invertases of revertant D10-ER1 was ruled out by appropriate crosses. We conclude that SUC1 is a structural gene that codes for both the large and the small forms of invertase and suggest that SUC2 through SUC5 are structural genes as well.

SUC1 gene of Saccharomyces: a structural gene for the large (glycoprotein) and small (carbohydrate-free) forms of invertase.

Saccharomyces cerevisiae revertant strain D10-ER1 has been shown to contain thermosensitive forms of the large (glycoprotein) and small (carbohydrate-free) invertases and a very low level of the small enzyme, along with a wild-type level of the large form (T. Mizunaga et al., Mol. Cell. Biol. 1:460-468, 1981). These characteristics cosegregated in crosses of the revertant strain with wild-type sucrose-fermenting (SUC1) or nonfermenting (suc0) strains. In addition, there is tight linkage between sucrose and maltose fermentation in revertant D10-ER1 (characteristic of the SUC1 and MAL1 genes). From this we infer that a single reversion event is responsible for the several changes observed in D10-ER1, and that this mutation maps within or very close to the SUC1 gene present in the ancestor strain 4059-358D. The revertant SUC1 allele in D10-ER1 (termed SUC1-R1) was expressed independently of the wild-type SUC1 gene when both were present in diploid cells. Diploids carrying only the wild-type or the mutant genes synthesized invertases with the characteristics of the parental Suc+ haploids. The possibility that a modifier gene was responsible for the alterations in the invertases of revertant D10-ER1 was ruled out by appropriate crosses. We conclude that SUC1 is a structural gene that codes for both the large and the small forms of invertase and suggest that SUC2 through SUC5 are structural genes as well.

Genetic evidence for a silent SUC gene in yeast.

The SUC genes (SUC1-SUC7) of Saccharomyces are a family of genes that are dispersed in the yeast genome. A SUC+ allele at any locus confers the ability to produce the enzyme invertase and, thus, to ferment sucrose. Most yeast strains do not carry SUC+ alleles at all possible SUC loci. We have investigated the naturally occurring negative (suc0) alleles present at SUC loci with the aim of distinguishing between two possible models for the structure of suc0 alleles: (1) suc0 alleles correspond to a simple absence of SUC genetic information; (2) suc0 alleles are "silent" SUC genes that either produce a defective product or are not expressed. To facilitate these studies, sucrose-nonfermenting strains were constructed that are congenic to S. cerevisiae strain S288C (SUC2+), but carry at the SUC2 locus the naturally occurring negative allele, suc2(0), of strain FL100 (Lacroute 1968). These strains were used to study the genetic properties of the suc2(0) allele of FL100 and the suc0 alleles (suc1(0), suc3(0), etc.) of S288C. The suc2(0) allele was shown to revert to an active Suc+ state and to provide functional information at three points in the SUC2 gene in recombination experiments; this suc2(0) gene thus appears to be a "silent" gene. Similar tests for silent SUC genes in S288C (corresponding to loci other than SUC2) failed to reveal any additional silent genes.

Temperature-sensitive forms of large and small invertase in a mutant derived from a SUC1 strain of Saccharomyces cerevisiae.

Mutagenesis of the sucrose-fermenting (SUC1) Saccharomyces cerevisiae strain 4059-358D yielded an invertase-negative mutant (D10). Subsequent mutagenic treatment of D10 gave a sucrose-fermenting revertant (D10-ER1) that contained the same amount of large (mannoprotein) invertase as strain 4059-358D but only trace amounts of the smaller intracellular nonglycosylated enzyme. Limited genetic evidence indicated that the mutations in D10 and D10-ER1 are allelic to the SUC1 gene. The large invertases from D10-ER1 and 4059-358D were purified and compared. The two enzymes have similar specific activity and Km for sucrose, cross-react immunologically, and show the same subunit molecular weight after removal of the carbohydrate with endo-beta-N-acetylglucosaminidae H. They differ in that the large enzyme from the revertant is rapidly inactivated at 55 degrees C, whereas that from the parent is relatively stable at 65 degrees C. The small invertase in extracts of D10-ER1 is also heat sensitive as compared to the small enzyme from the original parent strain. The low level of small invertase in mutant D10-ER1 may reflect increased intracellular degradation of this heat-labile form. In several crosses of D10-ER1 with strains carrying the SUC1 or SUC3 genes, the temperature sensitivity of the large and small invertases and the low cellular level of small invertase appeared to cosegregate. These findings are evidence that SUC1 is a structural gene for invertase and that both large and small forms are encoded by a single gene. A detailed genetic analysis is presented in a companion paper.

Mutants of yeast defective in sucrose utilization.

Utilization of sucrose as a source of carbon and energy in yeast (Saccharomyces) is controlled by the classical SUC genes, which confer the ability to produce the sucrose-degrading enzyme invertase (Mortimer and Hawthorne 1969). Mutants of S. cerevisiae strain S288C (SUC2+) unable to grow anaerobically on sucrose, but still able to use glucose, were isolated. Two major complementation groups were identified: twenty-four recessive mutations at the SUC2 locus (suc2-); and five recessive mutations defining a new locus, SNF1 (for sucrose nonfermenting), essential for sucrose utilization. Two minor complementation groups, each comprising a single member with a leaky sucrose-nonfermenting phenotype, were also identified. The Suc2 mutations isolated include four suppressible amber mutations and five mutations apparently exhibiting intragenic complementation; complementation analysis and mitotic mapping studies indicated that all of the suc2 mutations are alleles of a single gene. These results suggest that SUC2 encodes a protein, probably a dimer or multimer. No invertase activity was detected in suc2 probably a dimer or multimer. No invertase activity was detected in suc2 mutants,--The SNF1 locus is not tightly linked to SUC2. The snf1 mutations were found to be pleiotropic, preventing sucrose utilization by SUC2+ and SUC7+ strains, and also preventing utilization of galactose, maltose and several nonfermentable carbon sources. Although snf1 mutants thus display a petite phenotype, classic petite mutations do not interfere with utilization of sucrose, galactose or maltose. A common feature of all the carbon utilization systems affected by SNF1 is that all are regulated by glucose repression. The snf1 mutants were found to produce the constitutive nonglycosylated form of invertase, but failed to produce the glucose-repressible, glycosylated, secreted invertase. This failure cannot be attributed to a general defect in production of glycosylated and secreted proteins because synthesis of acid phosphatase, a glycosylated secreted protein not subject to glucose repression, was not affected by snf1 mutations. These findings suggest that the SNF1 locus is involved in the regulation of gene expression by glucose repression.

SUC genes of yeast: a dispersed gene family.

SUC genes of yeast: a dispersed gene family.

The structural genes of internal invertases in Saccharomyces cerevisiae

Polyacrylamide gel electrophoresis (without SDS) of invertases from strains each carrying only one of the five known SUC-genes revealed differences in mobility of the internal enzymes. SUC1 invertase moved distinctly slower than the invertases formed in the presence of genes SUC2 to SUC5. Three bands of internal invertase activity were found in diploids carrying both SUC1 (slow invertase) and one of the other SUC-genes (fast invertases). Tetrad analysis of such diploids yielded haploids which showed the same three bands if they carried SUC1 in combination with another SUC gene. A gene dosage effect was observed in relation to invertase activity in haploid strains with only gene SUC1 or only SUC4 on one hand, and both genes on the other hand. A sucrose non-fermenting and invertase negative strain with mutant allele suc3-3 of gene SUC3 (fast invertase) was crossed with SUC1. The heterozygous diploid and the recombinant haploids (SUC1 suc3-3) showed two bands in the region of the internal invertase: a slow SUC1 band and a second band corresponding to the intermediate band of SUC1-SUC3 strains. The intermediate band in SUC1 suc3-3 strains is considered as a hybrid consisting of an active SUC1-monomer and an inactive suc3-mutant monomer. Formation of such hybrid bands was taken as evidence for the structural nature of SUC-genes.

AN ACETATE-REQUIRING MUTANT STRAIN OF <i>NEUROSPORA</i> <i>CRASSA</i> DUE TO A POSSIBLE PARACENTRIC INVERSION

Acetate-requiring mutant strain 163 was investigated and compared with suc mutants. Growth and enzyme characteristics of the strain are similar to those of suc mutants. It is probable from genetic analyses that strain 163 carries a paracentric inversion near the mating type locus, between leu-4 and suc genes on the left arm of the first linkage group. The gene suc seems to be separated into two parts by the inversion, and this may be the reason for the acetate requirement and the similarity of strain 163 to suc mutants. Revertants were obtained from strain 163. All six revertants proved to be due to suppressor mutations. At least two different suppressor genes are involved. All the revertants produced spheroplasts and yeast-like morphology when they were cultivated with vigorous aeration in medium without acetate, while they showed normal mycelial structure in medium with acetate.

Amber mutants of the -ketoglutarate dehydrogenase gene of Escherichia coli K12.

SUMMARY Of 21 suc mutants of Escherichia coli k12 five were shown to be amber mutants by their sensitivity to amber suppressors. In addition to lacking activity for the overall Α-ketoglutarate dehydrogenase complex and the Α-ketoglutarate dehydrogenase component (E1), the amber mutants had little or no dihydrolipoyl trans-succinylase (E2) and 20 to 30% less dihydrolipoyl dehydrogenase (E3) than the parental strain. In common with non-suppressible suc mutants they also had reduced amounts of succinylCoA synthetase and succinate dehydrogenase. Fine structure mapping by Pi-transduction showed that some of the suc-amber sites were located in the sucA gene. It is concluded that expression of the suc region is polarized from sucA to sucB, i.e. synthesis of the E1 component precedes that of the E2 component. Double amber mutants with lesions in the pyruvate and Α-ketoglutarate dehydrogenase genes (aceE, sucA) were constructed but their E3 activity was never less than 30% of that of the parental strain. The results are consistent with the existence of separate genes for the dihydrolipoyl dehydrogenase component of the pyruvate and Α-ketoglutarate dehydrogenase complexes, but other possibilities could not be ruled out. Further studies on the positions of the four closely-linked tricarboxylic acid cycle genes specifying citrate synthase (gltA), succinate dehydrogenase (sdh) and two components of the Α-ketoglutarate dehydrogenase complex (sucA and sucB) indicate the relative order: gltA ‥sdh ‥sucA.sucB ‥tolII …gal.