The structural genes of internal invertases in Saccharomyces cerevisiae.

Summary 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.     

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.     

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.     

Comparative molecular-genetic analysis of the beta-fructosidases in yeast Saccharomyces

To infer the molecular evolution of polymeric beta-fructosidase SUC genes of the yeast Saccharomyces, we have cloned and sequenced a new SUC gene from S. cariocanus and determined the sequence similarity of beta-fructosidases within the genus Saccharomyces. The proteins of Saccharomyces cerevisiae and its five sibling species (S. bayanus, S. cariocanus, S. kudriavzevii, S. mikatae, S. paradoxus) have high degree of identity - 90-97%. The invertase of S. bayanus is the most divergent among the proteins studied. The data obtained indicated that the yeast invertases are highly conservative. In the coding regions of the SUC genes the pyrimidine transitions were the most abundant event due to silent changes mainly in the third codon position. There is only one, probably, non-telomeric SUC gene in each of the Saccharomyces species. In S. cerevisiae, S. bayanus, S. kudriavzevii, S. mikatae and S. paradoxus the SUC gene have been mapped on chromosome IX, whereas in S. cariocanus this gene is located in chromosone XV, in the position of translocation.     

Arabidopsis SUC1 loads the phloem in suc2 mutants when expressed from the SUC2 promoter

Active loading of sucrose into phloem companion cells (CCs) is an essential process in apoplastic loaders, such as Arabidopsis or tobacco (Nicotiana sp.), and is even used by symplastic loaders such as melon (Cucumis melo) under certain stress conditions. Reduction of the amount or complete removal of the transporters catalysing this transport step results in severe developmental defects. Here we present analyses of two Arabidopsis lines, suc2-4 and suc2-5, that carry a null allele of the SUC2 gene which encodes the Arabidopsis phloem loader. These lines were complemented with constructs expressing either the Arabidopsis SUC1 or the Ustilago maydis srt1 cDNA from the SUC2 promoter. Both SUC1 and Srt1 are energy-dependent sucrose/H(+) symporters and differ in specific kinetic properties from the SUC2 protein. Transgene expression was confirmed by RT-PCRs, the subcellular localization of Srt1 in planta with an Srt1-RFP fusion, and the correct CC-specific localization of the recombinant proteins by immunolocalization with anti-Srt1 and anti-SUC1 antisera. The transport capacity of Srt1 was studied in Srt1-GFP expressing Arabidopsis protoplasts. Although both proteins were found exclusively in CCs, only SUC1 complemented the developmental defects of suc2-4 and suc2-5 mutants. As SUC1 and Srt1 are well characterized, this result provides an insight into the properties that are essential for sucrose transporters to load the phloem successfully.     

Molecular analysis of SNF2 and SNF5, genes required for expression of glucose-repressible genes in Saccharomyces cerevisiae.

The SNF2 and SNF5 genes are required for derepression of SUC2 and other glucose-repressible genes of Saccharomyces cerevisiae in response to glucose deprivation. Previous genetic evidence suggested that SNF2 and SNF5 have functionally related roles. We cloned both genes by complementation and showed that the cloned DNA was tightly linked to the corresponding chromosomal locus. Both genes in multiple copy complemented only the cognate snf mutation. The SNF2 gene encodes a 5.7-kilobase RNA, and the SNF5 gene encodes a 3-kilobase RNA. Both RNAs contained poly(A) and were present in low abundance. Neither was regulated by glucose repression, and the level of SNF2 RNA was not dependent on SNF5 function or vice versa. Disruption of either gene at its chromosomal locus still allowed low-level derepression of secreted invertase activity, suggesting that these genes are required for high-level expression but are not directly involved in regulation. Further evidence was the finding that snf2 and snf5 mutants failed to derepress acid phosphatase, which is not regulated by glucose repression. The SNF2 and SNF5 functions were required for derepression of SUC2 mRNA.     

New Snf Genes, Gal11 and Grr1 Affect Suc2 Expression in Saccharomyces Cerevisiae

To identify new genes required for depression of the SUC2 (invertase) gene in Saccharomyces cerevisiae, we have isolated mutants with defects in raffinose utilization. In addition to mutations in SUC2 and previously identified SNF genes, we recovered recessive mutations that define four new complementation groups, designated snf7 through snf10. These mutations cause defects in the derepression of SUC2 in response to glucose limitation. We also recovered five alleles of gal11 and showed that a gal11 null mutation decreases SUC2 expression to 30% of the wild-type level. Finally, one of the mutants carries a grr1 allele that converts SUC2 from a glucose-inducible gene.     

Null mutations in the SNF3 gene of Saccharomyces cerevisiae cause a different phenotype than do previously isolated missense mutations.

Missense mutations in the SNF3 gene of Saccharomyces cerevisiae were previously found to cause defects in both glucose repression and derepression of the SUC2 (invertase) gene. In addition, the growth properties of snf3 mutants suggested that they were defective in uptake of glucose and fructose. We have cloned the SNF3 gene by complementation and demonstrated linkage of the cloned DNA to the chromosomal SNF3 locus. The gene encodes a 3-kilobase poly(A)-containing RNA, which was fivefold more abundant in cells deprived of glucose. The SNF3 gene was disrupted at its chromosomal locus by several methods to create null mutations. Disruption resulted in growth phenotypes consistent with a defect in glucose uptake. Surprisingly, gene disruption did not cause aberrant regulation of SUC2 expression. We discuss possible mechanisms by which abnormal SNF3 gene products encoded by missense alleles could perturb regulatory functions.     

SUCROSE TRANSPORTER 5 supplies Arabidopsis embryos with biotin and affects triacylglycerol accumulation

The Arabidopsis SUC5 protein represents a classical sucrose/H⁺ symporter. Functional analyses previously revealed that SUC5 also transports biotin, an essential co‐factor for fatty acid synthesis. However, evidence for a dual role in transport of the structurally unrelated compounds sucrose and biotin in plants was lacking. Here we show that SUC5 localizes to the plasma membrane, and that the SUC5 gene is expressed in developing embryos, confirming the role of the SUC5 protein as substrate carrier across apoplastic barriers in seeds. We show that transport of biotin but not of sucrose across these barriers is impaired in suc5 mutant embryos. In addition, we show that SUC5 is essential for the delivery of biotin into the embryo of biotin biosynthesis‐defective mutants (bio1 and bio2). We compared embryo and seedling development as well as triacylglycerol accumulation and fatty acid composition in seeds of single mutants (suc5, bio1 or bio2), double mutants (suc5 bio1 and suc5 bio2) and wild‐type plants. Although suc5 mutants were like the wild‐type, bio1 and bio2 mutants showed developmental defects and reduced triacylglycerol contents. In suc5 bio1 and suc5 bio2 double mutants, developmental defects were severely increased and the triacylglycerol content was reduced to a greater extent in comparison to the single mutants. Supplementation with externally applied biotin helped to reduce symptoms in both single and double mutants, but the efficacy of supplementation was significantly lower in double than in single mutants, showing that transport of biotin into the embryo is lower in the absence of SUC5.     

Genetic and genomic evidence that sucrose is a global regulator of plant responses to phosphate starvation in Arabidopsis

Plants respond to phosphate (Pi) starvation by exhibiting a suite of developmental, biochemical, and physiological changes to cope with this nutritional stress. To understand the molecular mechanism underlying these responses, we isolated an Arabidopsis (Arabidopsis thaliana) mutant, hypersensitive to phosphate starvation1 (hps1), which has enhanced sensitivity in almost all aspects of plant responses to Pi starvation. Molecular and genetic analyses indicated that the mutant phenotype is caused by overexpression of the SUCROSE TRANSPORTER2 (SUC2) gene. As a consequence, hps1 has a high level of sucrose (Suc) in both its shoot and root tissues. Overexpression of SUC2 or its closely related family members SUC1 and SUC5 in wild-type plants recapitulates the phenotype of hps1. In contrast, the disruption of SUC2 functions greatly inhibits plant responses to Pi starvation. Microarray analysis further indicated that 73% of the genes that are induced by Pi starvation in wild-type plants can be induced by elevated levels of Suc in hps1 mutants, even when they are grown under Pi-sufficient conditions. These genes include several important Pi signaling components and those that are directly involved in Pi transport, mobilization, and distribution between shoot and root. Interestingly, Suc and low-Pi signals appear to interact with each other both synergistically and antagonistically in regulating gene expression. Our genetic and genomic studies provide compelling evidence that Suc is a global regulator of plant responses to Pi starvation. This finding will help to further elucidate the signaling mechanism that controls plant responses to this particular nutritional stress.     

Genes Affecting the Regulation of SUC2 Gene Expression by Glucose Repression in SACCHAROMYCES CEREVISIAE

Mutants of Saccharomyces cerevisiae with defects in sucrose or raffinose fermentation were isolated. In addition to mutations in the SUC2 structural gene for invertase, we recovered 18 recessive mutations that affected the regulation of invertase synthesis by glucose repression. These mutations included five new snf1 (sucrose nonfermenting) alleles and also defined five new complementation groups, designated snf2, snf3, snf4, snf5, and snf6. The snf2, snf4, and snf5 mutants produced little or no secreted invertase under derepressing conditions and were pleiotropically defective in galactose and glycerol utilization, which are both regulated by glucose repression. The snf6 mutant produced low levels of secreted invertase under derepressing conditions, and no pleiotropy was detected. The snf3 mutants derepressed secreted invertase to 10-35% the wild-type level but grew less well on sucrose than expected from their invertase activity; in addition, snf3 mutants synthesized some invertase under glucose-repressing conditions.--We examined the interactions between the different snf mutations and ssn6, a mutation causing constitutive (glucose-insensitive) high-level invertase synthesis that was previously isolated as a suppressor of snf1. The ssn6 mutation completely suppressed the defects in derepression of invertase conferred by snf1, snf3, snf4 and snf6, and each double mutant showed the constitutivity for invertase typical of ssn6 single mutants. In contrast, snf2 ssn6 and snf5 ssn6 strains produced only moderate levels of invertase under derepressing conditions and very low levels under repressing conditions. These findings suggest roles for the SNF1 through SNF6 and SSN6 genes in the regulation of SUC2 gene expression by glucose repression.     

Relationship of large and small invertases in Saccharomyces: mutant selectively deficient in small invertase.

A mutant strain of Saccharomyces cerevisiae (D10-ER1) has been isolated after a two-step mutagenesis of strain 4059-358D (SUC 1) using ethyl methane sulfonate. Cells of this new strain produced a level of total invertase equaling that of 4059 but contained only trace amounts of the small, internal, aglycan form of the enzyme (less than 0.1% of total in D10-ER1 compared with 6% in 4059). When D10.ER1 was crossed with an invertase-hyperproducing strain dgr3 (SUC3), progeny were isolated (HZ400-5A and HZ400-2C) in which levels of total invertase had at least quadrupled. The percentage of small invertase, however, remained insignificant. Levels of small invertase in strain HZ400-5A were determined by affinity chromatography on conconavalin A-Sepharose, gel permeation chromatography, and isopycnic centrifugation in CsCl. The large invertase of the SUC1 yeasts described here was found to contain a form apparently greater in size than the large invertase of the SUC2 strain FH4C; this probably reflects a higher content of carbohydrate. The overall results of this study do not support a direct structural relationship between large and small invertases. The implications on invertase biosynthesis and structure are discussed.     

The amino terminus of the yeast F1-ATPase beta-subunit precursor functions as a mitochondrial import signal

The ATP2 gene of Saccharomyces cerevisiae codes for the cytoplasmically synthesized beta-subunit protein of the mitochondrial F1-ATPase. To define the amino acid sequence determinants necessary for the in vivo targeting and import of this protein into mitochondria, we have constructed gene fusions between the ATP2 gene and either the Escherichia coli lacZ gene or the S. cerevisiae SUC2 gene (which codes for invertase). The ATP2-lacZ and ATP2-SUC2 gene fusions code for hybrid proteins that are efficiently targeted to yeast mitochondria in vivo. The mitochondrially associated hybrid proteins fractionate with the inner mitochondrial membrane and are resistant to proteinase digestion in the isolated organelle. Results obtained with the gene fusions and with targeting-defective ATP2 deletion mutants provide evidence that the amino-terminal 27 amino acids of the beta-subunit protein precursor are sufficient to direct both specific sorting of this protein to yeast mitochondria and its import into the organelle. Also, we have observed that certain of the mitochondrially associated Atp2-LacZ and Atp2-Suc2 hybrid proteins confer a novel respiration-defective phenotype to yeast cells.     

酵母SUC2基因上游顺序对其表达的影响

从SUC2基因上游约—900bp向起始密码进行系列缺失。将带有这种缺失上游区的SUC2基因插入多拷贝质粒,并转化进不产蔗糖酶的酵母细胞。测定了这些缺失株表达蔗糖酶的数量。结果表明:在葡萄糖阻遏条件下,SUC2上游区缺失从-636bp到-179bp的不同细胞,糖基化蔗糖酶的表达量逐渐升高。和野生型相比,SUC2上游区缺失到-223bp和-179bp的细胞糖基化蔗糖酶量增加100倍以上。在葡萄糖去阻遏条件下,SUC2上游缺失从-395bp到-179bp的不同细胞,糖基化蔗糖酶的表达量只显示微弱的去阻遏效应。缺失末端达-89bp和-41bp的细胞只表达很少的糖基化蔗糖酶,但是非糖基化蔗糖酶的表达量明显增加。     

Genetic control of invertase formation in Saccharomyces cerevisiae. II. Isolation and characterization of mutants conferring invertase hyperproduction in strain EK-6B carrying the SUC3 gene.

Summary Invertase formation in the yeast Saccharomyces cerevisiae is subject to repression by hexoses in the growth medium. Mutagen-induced (ethyl methanesulfonate or N-methyl-N'-nitro-nitrosoguanidine) invertase hyperproducer mutants have been derived from the SUC3 MAL3 strain EK-6B by selecting for their ability to grow on media containing the sugar raffinose plus 2-deoxy-D-glucose (2DG). Raffinose like sucrose is a -fructoside which can be hydrolyzed by yeast invertase (-fructofuranoside fructohydrolase). These mutants, designated dgr, produce higher levels of invertase (-glucosidase levels are also elevated but to a lesser extent) under conditions normally repressing invertase biosynthesis in the parent. Invertases of mutants dgr2 and dgr3 are indistinguishable from that of EK-6B with respect to their Km's for sucrose and thermal labilities. Genetic studies revealed that dgr2 and dgr3 are recessive and unlinked to the SUC3 gene.