The secreted form of invertase in Saccharomyces cerevisiae is synthesized from mRNA encoding a signal sequence.

The SUC2 gene of Saccharomyces cerevisiae encodes two differently regulated mRNAs (1.8 and 1.9 kilobases) that differ at their 5' ends. The larger RNA encodes a secreted, glycosylated form of invertase and the smaller RNA encodes an intracellular, nonglycosylated form. We have determined the nucleotide sequence of the amino-terminal coding region of the SUC2 gene and its upstream flanking region and have mapped the 5' ends of the SUC2 mRNAs relative to the DNA sequence. The 1.9-kilobase RNA contains a signal peptide coding sequence and presumably encodes a precursor to secreted invertase. The 1.8-kilobase RNA does not include the complete coding sequence for the signal peptide. The nucleotide sequence data prove that SUC2 is a structural gene for invertase, and translation of the coding information provides the complete amino acid sequence of an S. cerevisiae signal peptide.     

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.     

Invertase beta-galactosidase hybrid proteins fail to be transported from the endoplasmic reticulum in Saccharomyces cerevisiae.

The yeast SUC2 gene codes for the secreted enzyme invertase. A series of 16 different-sized gene fusions have been constructed between this yeast gene and the Escherichia coli lacZ gene, which codes for the cytoplasmic enzyme beta-galactosidase. Various amounts of SUC2 NH2-terminal coding sequence have been fused in frame to a constant COOH-terminal coding segment of the lacZ gene, resulting in the synthesis of hybrid invertase-beta-galactosidase proteins in Saccharomyces cerevisiae. The hybrid proteins exhibit beta-galactosidase activity, and they are recognized specifically by antisera directed against either invertase or beta-galactosidase. Expression of beta-galactosidase activity is regulated in a manner similar to that observed for invertase activity expressed from a wild-type SUC2 gene: repressed in high-glucose medium and derepressed in low-glucose medium. Unlike wild-type invertase, however, the invertase-beta-galactosidase hybrid proteins are not secreted. Rather, they appear to remain trapped at a very early stage of secretory protein transit: insertion into the endoplasmic reticulum (ER). The hybrid proteins appear only to have undergone core glycosylation, an ER process, and do not receive the additional glycosyl modifications that take place in the Golgi complex. Even those hybrid proteins containing only a short segment of invertase sequences at the NH2 terminus are glycosylated, suggesting that no extensive folding of the invertase polypeptide is required before initiation of transmembrane transfer. beta-Galactosidase activity expressed by the SUC2-lacZ gene fusions cofractionates on Percoll density gradients with ER marker enzymes and not with other organelles. In addition, the hybrid proteins are not accessible to cell-surface labeling by 125I. Accumulation of the invertase-beta-galactosidase hybrid proteins within the ER does not appear to confer a growth-defective phenotype to yeast cells. In this location, however, the hybrid proteins and the beta-galactosidase activity they exhibit could provide a useful biochemical tag for yeast ER membranes.     

Comparison of two yeast invertase genes: conservation of the upstream regulatory region.

The yeast genome contains a dispersed family of invertase structural genes (SUC1-SUC5, SUC7). Five of these genes are located very close to telomeres and are flanked by large regions of homologous sequence; recombination between telomeres could account for the dispersal of these SUC genes to different chromosomes. The SUC2 locus, in contrast, is not near a telomere and does not share large regions of flanking homology with the other loci. We examine here the relationship between SUC2 and one of the telomeric genes, SUC7. Sequence comparison revealed homology extending from about position -624 to +1791, which is close to the end of the mRNA. The 5' noncoding sequence includes two highly conserved regions: the region between -140 and +1, which contains the TATA box and presumably other promoter elements, and a second region extending from -508 to -400, which corresponds to the upstream regulatory region.     

Fine analysis of the chromatin structure of the yeast SUC2 gene and of its changes upon derepression. Comparison between the chromosomal and plasmid-inserted genes.

Micrococcal nuclease digestion has been used to investigate some fine details of the chromatin structure of the yeast SUC2 gene for invertase. Precisely positioned nucleosomes have been found on a 2 kb sequence from the 3' non-coding region, and four nucleosomes also seem to occupy fixed positions on the 5' flank. Eleven nucleosomes lie on the coding region, although their positioning is not as precise as in the flanks. When the gene is derepressed, these latter nucleosomes adopt a more open conformation and so do two of the nucleosomes positioned on the 5' flank. A dramatic change occurs in the 3' flank, whose involvement in the structural transitions of chromatin upon gene activation is postulated. All the observed features are conserved when the gene is inserted in either a single copy centromeric plasmid or in a multicopy, 2 micron circle-based plasmid.     

Invertase signal and mature sequence substitutions that delay intercompartmental transport of active enzyme

The role of structural signals in intercompartmental transport has been addressed by the isolation of yeast invertase (SUC2) mutations that cause intracellular accumulation of active enzyme. Two mutations that delay transport of core-glycosylated invertase, but not acid phosphatase, have been mapped in the 5' coding region of SUC2. Both mutations reduce specifically the transport of invertase to a compartment, presumably in the Golgi body, where outer chain carbohydrate is added. Subsequent transport to the cell surface is not similarly delayed. One mutation (SUC2-s1) converts an ala codon to val at position -1 in the signal peptide; the other (SUC2-s2) changes a thr to an ile at position +64 in the mature protein. Mutation s1 results in about a 50-fold reduced rate of invertase transport to the Golgi body which is attributable to defective signal peptide cleavage. While peptide cleavage normally occurs at an ala-ser bond, the s1 mutant form is processed slowly at the adjacent ser-met position giving rise to mature invertase with an N-terminal met residue. s2 mutant invertase is transported about sevenfold more slowly than normal, with no delay in signal peptide cleavage, and no detectable abnormal physical property of the enzyme. This substitution may interfere with the interaction of invertase and a receptor that facilitates transport to the Golgi body.     

Nucleotide sequence of the sucrase gene of Bacillus subtilis

The sucrase gene (sacA) and part of the sacP locus, which corresponds to a membrane component of the phosphotransferase system (PTS) of sucrose transport of Bacillus subtilis, were previously cloned on a 2.1-kb EcoRI DNA fragment. Genes sacA and sacP were localized on this DNA fragment and the nucleotide sequence of the 2.1-kb DNA fragment was determined. A 1440-bp open reading frame (480 codons) was identified coding for a deduced polypeptide of Mr54827, which corresponds to that of purified sucrase. The amino acid sequence shares homology with that of yeast invertase (SUC2 gene product). The sacA gene and the preceding sacP gene seem to belong to the same operon.     

Short repeated elements in the upstream regulatory region of the SUC2 gene of Saccharomyces cerevisiae.

Expression of secreted invertase from the SUC2 gene is regulated by carbon catabolite repression. Previously, an upstream regulatory region that is required for derepression of secreted invertase was identified and shown to confer glucose-repressible expression to the heterologous promoter of a LEU2-lacZ fusion. In this paper we show that tandem copies of a 32-base pair (bp) sequence from the upstream regulatory region activate expression of the same LEU2-lacZ fusion. The level of expression increased with the number of copies of the element, but was independent of their orientation; the expression from constructions containing four copies of the sequence was only twofold lower than that when the entire SUC2 upstream regulatory region was present. This activation was not significantly glucose repressible. The 32-bp sequence includes a 7-bp motif with the consensus sequence (A/C)(A/G)GAAAT that is repeated at five sites within the upstream regulatory region. Genetic evidence supporting the functional significance of this repeated motif was obtained by pseudoreversion of a SUC2 deletion mutant lacking part of the upstream region, including two copies of the 7-bp element. In three of five pseudorevertants, the mutations that restored high-level SUC2 expression altered one of the remaining copies of the 7-bp element.     

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.     

Effects of temperature and cycloheximide on secretion of cloned invertase from recombinant Saccharomyces cerevisiae

The effects of temperature on the kinetics and efficiency of secretion of cloned invertase were investigated in a recombinant yeast system. This system consisted of the baker's yeast Saccharomyces cerevisiae (SEY2102) transformed with the 2mu-based plasmid pBR58 which contains the entire SUC2 gene including the promoter, signal sequence, and structural gene. The recombinant yeast produces the naturally secreted yeast enzyme invertase. In transition experiments done at temperatures ranging from 25 degrees to 45 degrees C, the maximum invertase level and secretion rate exhibited maxima of 5.5 U/mL . OD and 4.6 U/mL . OD per hour, respectively, at 35 degrees C. Experiments involving the use of cycloheximide showed that it took approximately 15 min for secreted invertase to move through the secretion pathway, which held 0.4 U/mL . OD of specific activity. (c) 1995 John Wiley & Sons, Inc.