Internal Invertase Research Articles

The Saccharomyces cerevisiae vacuolar acid trehalase is targeted at the cell surface for its physiological function

Previous studies in the yeast Saccharomyces cerevisiae have proposed a vacuolar localization for Ath1, which is difficult to reconcile with its ability to hydrolyze exogenous trehalose. We used fluorescent microscopy to show that the red fluorescent protein mCherry fused to the C-terminus of Ath1, although mostly localized in the vacuole, was also targeted to the cell surface. Also, hybrid Ath1 truncates fused at their C-terminus with the yeast internal invertase revealed that a 131 amino acid N-terminal fragment of Ath1was sufficient to target the fusion protein to the cell surface, enabling growth of the suc2Delta mutant on sucrose. The unique transmembrane domain appeared to be indispensable for the production of a functional Ath1, and its removal abrogated invertase secretion and growth on sucrose. Finally, the physiological significance of the cell-surface localization of Ath1 was established by showing that fusion of the signal peptide of invertase to N-terminal truncated Ath1 allowed the ath1Delta mutant to grow on trehalose, whereas the signal sequence of the vacuolar-targeted Pep4 constrained Ath1 in the vacuole and prevented growth of this mutant on trehalose. Use of trafficking mutants that impaired Ath1 delivery to the vacuole abrogated neither its activity nor its growth on exogenous trehalose.

Genetic and proteomic evidences support the localization of yeast enolase in the cell surface

Although enolase, other glycolytic enzymes, and a variety of cytoplasmic proteins lacking an N-terminal secretion signal have been widely described as located at the cell surface in yeast and in mammalian cells, their presence in this external location is still controversial. Here, we report that different experimental approaches (genetics, cellular biology and proteomics) show that yeast enolase can reach the cell surface and describe the protein regions involved in its cell surface targeting. Hybrid enolase truncates, fused at their C terminus with the yeast internal invertase or green fluorescent protein (GFP) as reporter proteins, proved that the 169 N-terminal amino acids are sufficient to target the protein to the cell surface. Furthermore, the enolase-GFP fusion co-localized with a plasma membrane marker. Enolase was also identified among membrane proteins obtained by a purification protocol that includes sodium carbonate to prevent cytoplasmic contamination. These proteins were analyzed by SDS-PAGE, trypsin digestion and LC-MS/MS for peptide identification. Elongation factors, mitochondrial membrane proteins and a mannosyltransferase involved in cell wall mannan biosynthesis were also identified in this fraction.

Starvation and temperature upshift cause an increase in the enzymatically active cell wall-associated glyceraldehyde-3-phosphate dehydrogenase protein in yeast.

The cell wall-associated glyceraldehyde-3-phosphate dehydrogenase (cwGAPDH) activity in Saccharomyces cerevisiae increases (two- to 10-fold, depending on the strain) in response to starvation and temperature upshift. Assays using transformants carrying pTDH, a yeast centromer derivative plasmid containing the Candida albicans TDH3 gene (encoding GAPDH) fused in frame with the yeast SUC2-coding region for internal invertase, showed that starvation and/or temperature upshift result in a similar increase in both cwGAPDH and cell wall-associated invertase activities. In addition, this incorporation of GAPDH protein into the cell wall in response to stress does not require (i) de novo protein synthesis, indicating that preexisting cytosolic enzyme is incorporated into the cell wall, (ii) nor the participation of the ubiquitin yeast stress response system, as no differences were observed between wild-type and polyubiquitin-depleted (Deltaubi4) strains.

Candida albicans TDH3 gene promotes secretion of internal invertase when expressed in Saccharomyces cerevisiae as a glyceraldehyde-3-phosphate dehydrogenase-invertase fusion protein.

We have checked the ability of the Candida albicans GAPDH polypeptide, which lacks a conventional N-terminal signal peptide, to reach the cell wall in Saccharomyces cerevisiae by using an intracellular form of the yeast invertase as a reporter protein. A hybrid TDH3-SUC2 gene containing the C. albicans TDH3 promoter sequences and a coding region encoding a fusion protein formed by the C. albicans GAPDH polypeptide, fused at its C-terminus with the yeast internal invertase, was constructed in a centromer derivative plasmid and transformed into a Suc(-) S. cerevisiae strain. Transformants displayed invertase activity measured in intact whole cells, and were able to grow on sucrose as the sole fermentable carbon source. Northern blot analysis with both TDH3 and SUC2 probes detected a single mRNA species of the expected size (about 2.7 kb), and Western immunoblot analysis of cell-free extracts, using a monoclonal antibody (mAb49) against a C. albicans GAPDH epitope, showed the presence of a 90 kDa polypeptide corresponding to the GAPDH-invertase fusion protein. This indicates that the TDH3 gene is able to direct part of the encoded gene product to the cell wall, and that any putative motifs for this targeting should be within the GAPDH amino acid sequence. Further analysis, using the same approach, of a panel of seven N- and C-terminal GAPDH truncates revealed that the region required for the cell wall targeting is located within the N-terminal half of the protein.

The vanadate-tolerant yeast Hansenula polymorpha undergoes cellular reorganization during growth in, and recovery from, the presence of vanadate.

When present at intracellular concentrations above micromolar, vanadate becomes toxic to most organisms. However, the yeast Hansenula polymorpha is able to grow on vanadate concentrations in the millimolar range, showing at the same time modifications in cellular ultrastructure and polyphosphate metabolism. Here, the development of the ultrastructural changes, and of vacuolar and secretory activities, during exponential growth on vanadate and upon a return to vanadate-free conditions was investigated. External invertase secretion was inhibited by vanadate, as shown by a decrease in external invertase activity, an intracellular accumulation of small vesicles and a cytoplasmic accumulation of internal invertase. An aberrant appearance of the cell wall and defects in cellular surface growth, possibly linked to defects in secretion, were also observed. However, inhibition of the secretory pathway was not complete since the activity of another secreted enzyme, exoglucanase, increased in the presence of vanadate. Growth on vanadate was also accompanied by an enhancement of vacuolar proteolysis, as indicated by an increase in carboxypeptidase Y activity. However, these modifications were all reversible upon return to vanadate-free conditions, with the normalization process being complex and involving new and dramatic ultrastructural changes and activation of an autophagic mechanism. This mechanism is involved in the elimination/resorption of the observed vanadate-induced aberrant cell structures and/or sites involved in vanadate accumulation, a necessary prerequisite for restoration of conventional ultrastructure and metabolic functions.

Effects of the expression of mammalian annexins in yeast secretory mutants.

The hypothesis that calcium-dependent membrane-binding proteins of the annexin family can influence intracellular membrane trafficking was tested by expressing five mammalian annexins in wild-type yeast cells (Saccharomyces cerevisiae) and in 13 yeast secretory (sec) mutants. Expression of human synexin (annexin VII) inhibited the growth of sec2, sec4 and sec15 mutants at a semi-permissive temperature. These three sec mutants are defective in the final step in the secretory pathway, the process of exocytosis. The inhibition of growth correlated with reduced viability and increased accumulation of internal invertase in these mutants when expressing synexin. Bovine endonexin (annexin IV) partially suppressed the growth defect of a sec2 mutant incubated at a semi-permissive temperature. Human synexin, human lipocortin (annexin I), and murine p68 (annexin VI) reduced the lag time associated with adaptation of sec2 mutants to galactose-containing medium. These interactions suggest that the annexins may influence specific steps in membrane trafficking associated with cell growth, secretion and plasma membrane remodelling.

Effect of oligosaccharides and chloride on the oligomeric structures of external, internal, and deglycosylated invertase

External invertase exists in an oligomeric equilibrium of dimer, tetramer, hexamer, and octamer, the concentrations of which vary with pH, time, and concentration of enzyme [Chu, F.K., Watorek, W., & Maley, F. (1983) Arch. Biochem. Biophys. 223, 543-555; Tammi, M., Ballou, L., Taylor, A., & Ballou, C.E. (1987) J. Biol. Chem. 262, 4395-4401]. To assess the influence of carbohydrate on this equilibrium, we investigated the self-association of external invertase (10 oligosaccharides per subunit), deglycosylated external invertase (2 oligosaccharides per subunit), and internal invertase (no carbohydrate) under various conditions. In addition, the effect of carbohydrate on the interaction of the subunits of these various invertases to form heterooligomers was studied. Chloride ion was found to promote subunit association in the various invertases irrespective of their glycosylation status. However, external invertase was less responsive to chloride ion relative to the internal and deglycosylated invertases. The higher oligomers of deglycosylated invertase were stable at 47 degrees C whereas those of external invertase dissociated rapidly into dimers, suggesting that the additional oligosaccharides in external invertase destabilize subunit interaction. Hybridization experiments with the various invertases showed that the dimers of internal invertase formed heterooligomers with either external or deglycosylated invertase. By contrast, the monomers of external and internal invertases formed their respective homodimers, but not heterodimers. These results suggest that the oligosaccharide content of invertase not only influences the extent of self-association but also affects heterooligomer formation.

Purification and partial characterization of external and internal invertases from two strains of trichosporon adeninovorans

AbstractWe investigated the extracellular and intracellular invertase activity of two Trichosporon adeninovorans strains. Both strains (SBUG 724 and CBS 2844) secrete one invertase into the medium. The external enzymes were purified by chromatography on hydroxylapatite. The molecular weight of the external invertase of SBUG 724 was found to be 650,000 and of CBS 2844 450,000. The internal invertases were separated by DEAE‐cellulose chromatography. The molecular weight of the enzyme from CBS 2844 was estimated to be 125,000. In the strain SBUG 724 we found two internal invertases (Mr 230,000 and 70,000). The activity of all invertases has similar properties. The pH optimum of the reaction was determined between 5.0 and 5.2 and the temperature optimum was 60 to 70°C. The Km value for sucrose was determined to be 71 to 83 mM and for raffinosc 27 to 36 mM.

Characterization of the glycosylation sites in yeast external invertase. I. N-linked oligosaccharide content of the individual sequons.

External invertase is the product of the SUC2 gene of Saccharomyces cerevisiae. The deduced sequence of this enzyme (Taussig, R., and Carlson, M. (1983) Nucleic Acid Res. 11, 1943-1954) reveals it to contain 14 potential N-linked glycosylation sites, or sequons, although only 9-10 appear to be glycosylated (Trimble, R. B., and Maley, F. (1977) J. Biol. Chem. 252, 4409-4412). To determine the location of the glycosylated sequons, external invertase was deglycosylated with endo-beta-acetylglucosaminidase H and its component peptides analyzed by both fast atom bombardment mass spectrometry (FABMS) and classical peptide isolation procedures. By use of the former technique most of the glucosamine-containing sequons could be located and by the latter sufficient amounts of small glucosamine-containing peptides were isolated to enable their quantitation. From the combined FABMS and glucosamine analyses, it was established that eight of the sequons in a subunit of invertase are either completely or almost completely glycosylated, while five others are glycosylated to the extent of about 50% or less. In the case of two overlapping sequons (4 and 5), which include Asn92-Asn93-Thr-Ser, only the first Asn was glycosylated. Thus, all but one of the sequons of external invertase are glycosylated to some extent, giving an appearance of only 9-10 N-linked oligosaccharides/subunit. The sequence identity of both external and internal invertase was verified by FABMS and by peptide sequence analysis. In only one site was an amino acid found to differ from that deduced from the DNA sequence of the SUC2 gene. This occurred at position 390 where a proline was found in place of alanine, which could result from a single base change in the triplet specifying the latter amino acid.

Secretion-Defective Mutations in the Signal Sequence for Saccharomyces cerevisiae Invertase

Nine mutations in the signal sequence region of the gene specifying the secreted Saccharomyces cerevisiae enzyme invertase were constructed in vitro. The consequences of these mutations were studied after returning the mutated genes to yeast cells. Short deletions and two extensive substitution mutations allowed normal expression and secretion of invertase. Other substitution mutations and longer deletions blocked the formation of extracellular invertase. Yeast cells carrying this second class of mutant gene expressed novel active internal forms of invertase that exhibited the following properties. The new internal proteins had the mobilities in denaturing gels expected of invertase polypeptides that had retained a defective signal sequence and were otherwise unmodified. The large increase in molecular weight characteristic of glycosylation was not seen. On nondenaturing gels the mutant enzymes were found as heterodimers with a normal form of invertase that is known to be cytoplasmic, showing that the mutant forms of the enzyme are assembled in the same compartment as the cytoplasmic enzyme. All of the mutant enzymes were soluble and not associated with the membrane components after fractionation of crude cell extracts on sucrose gradients. Therefore, these signal sequence mutations result in the production of active internal invertase that has lost the ability to enter the secretory pathway. This demonstrates that the signal sequence is required for the earliest steps in membrane translocation.

Secretion-defective mutations in the signal sequence for Saccharomyces cerevisiae invertase.

Nine mutations in the signal sequence region of the gene specifying the secreted Saccharomyces cerevisiae enzyme invertase were constructed in vitro. The consequences of these mutations were studied after returning the mutated genes to yeast cells. Short deletions and two extensive substitution mutations allowed normal expression and secretion of invertase. Other substitution mutations and longer deletions blocked the formation of extracellular invertase. Yeast cells carrying this second class of mutant gene expressed novel active internal forms of invertase that exhibited the following properties. The new internal proteins had the mobilities in denaturing gels expected of invertase polypeptides that had retained a defective signal sequence and were otherwise unmodified. The large increase in molecular weight characteristic of glycosylation was not seen. On nondenaturing gels the mutant enzymes were found as heterodimers with a normal form of invertase that is known to be cytoplasmic, showing that the mutant forms of the enzyme are assembled in the same compartment as the cytoplasmic enzyme. All of the mutant enzymes were soluble and not associated with the membrane components after fractionation of crude cell extracts on sucrose gradients. Therefore, these signal sequence mutations result in the production of active internal invertase that has lost the ability to enter the secretory pathway. This demonstrates that the signal sequence is required for the earliest steps in membrane translocation.

A silent gene allelicto yeast invertase structural gene SUC2 can be activated by mutation to direct the formation of a new type of invertase

Strains fromSaccharomyces cerevisiae unable to produce invertase (suc0) may revert to invertase formation by ultraviolet treatment or spontaneously at a lower rate, as a consequence of activation of a silent gene allelic to the SUC2 locus. However, the new internal protein is physically different from the internal invertase expressed by the SUC2 gene according to electrophoresis analysis.

Comparative properties of amplified external and internal invertase from the yeast SUC2 gene.

Saccharomyces cerevisiae external and internal invertases have been amplified by introducing the normal and modified SUC2 genes into yeast multicopy plasmids, which were then used to transform a yeast strain resistant to repression by glucose. Amino acid compositional analysis of these enzymes, in addition to end group sequencing, confirmed the DNA sequence data of Taussig and Carlson (Taussig, R., and Carlson, M. (1983) Nucleic Acids Res. 11, 1943-1954), indicating that both enzymes were encoded in the same gene. Comparison of the properties of carbohydrate-containing external invertase and its nonglycosylated internal form revealed that although the carbohydrate did not appear to influence the conformation of the peptide backbone, as determined by circular dichroism analyses, its presence considerably enhanced the ability of guanidine HCl-denatured external invertase to be renatured relative to internal invertase. The Mr of the internal enzymes was found to be greatly dependent on pH with the enzyme being a monomer at pH 9.4, a dimer at pH 8.3, and an apparent octamer at pH 4.9.

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.

A membrane-associated isozyme of invertase in yeast precursor of the external glycoprotein

An enzymatic test is described which allows the localization of yeast invertase activity directly on sodium dodecyl sulfate gels. When crude membrane fractions are prepared from Saccharomyces cerevisiae cells which are actively synthesizing external invertase, these membranes show an activity band on sodium dodecyl sulfate gels additional to the external and the internal invertase. In the soluble fraction this form was completely absent. It has a molecular weight of approximately 190 000 and was 50 000 smaller than the external invertase. It showed kinetic characteristics of a precursor of the external enzyme. Thus it appeared transiently, when yeast cells were shifted from a condition of non-synthesizing external invertase to one where the enzyme was synthesized. When the increase in the external enzyme slowed down after some time, the membrane-associated form almost completely disappeared. The addition of tunicamycin to yeast cells synthesizing external invertase inhibited further synthesis of the enzyme by 97%; also the formation of the membrane-associated form was strongly inhibited. The amount of it present before the addition of tunicamycin completely disappeared in the presence of the antibiotic. The precursor form, therefore, seems to possess already those carbohydrate parts which contain N-acetylglucosamine and are transferred via dolichyl phosphate-bound intermediates. The membrane-associated precursor amounts to less than 5% of the total invertase activity of a yeast cell.

Effects of proteolytic enzymes on invertase secretion in sphaeroplasts of Saccharomyces: inhibition by trypsin.

Exogenously added trypsin arrested invertase secretion by sphacroplasts of Saccharomyces strain 1016. The mechanism of inhibition is presumed due to attack on plasma membrane protein(s). Gross membrane damage by trypsin was not apparent, as evidence by the absence of leakage of intracellular alkaline phosphatase, after trypsin treatment. Trypsin treatment did induce an increased sensitivity to lysis, observed only when changes in osmotic pressure were made and fresh glucose added. While synthesis of invertase was eventually inhibited by trypsin, a greater than twofold increase in internal invertase was observed, due to complete inhibition of secretion. This is the first report of the uncoupling of synthesis and secretion in yeast.

A modified procedure for studying enzyme secretion in yeast sphaeroplasts: subcellular distribution of invertase

A significantly modified procedure for investigating enzyme secretion from yeast sphaeroplasts, and results from its application are described. Sphaeroplasts were derepressed for invertase biosynthesis in the presence of helicase and fractionated to reveal the distribution of high and low molecular weight forms of invertase. Secreted enzyme was found to be of high molecular weight, exclusively. Less than 10% of the total invertase activity was present in washed sphaeroplasts and of this, 43% was soluble, consisting of both high and low molecular weight forms of invertase. Washed membranes retained 32% of the internal invertase activity, and on solubilization with Triton X-100 the enzyme was found to be of an intermediate molecular weight. These results are consistent with the hypothesis that invertase is glycosylated at the plasma membrane.

Microheterogeneity in yeast invertase

Yeast external invertase (EC 3.2.1.26), a glycoenzyme consisting of equal parts by weight of protein and mannan, has been found to contain covalently bound phosphate. Three preparations (from two yeast strains) had mannose/PO 4 ratios of 31–35, equivalent to 24–27 PO 4 residues per mol of enzyme, while a fourth had only 7 PO 4 residues per mol. From one of the high-PO 4 enzymes, approx. 69% of the phosphorus was recovered as mannose 6-phosphate. No correlation was found between invertase activity and phosphorus content. The PO 4 contents of the invertases exceeded those of the cell wall mannans from the respective yeasts. Thus, contamination of the invertases by cell wall phosphomannan is unlikely. Electrofocusing of the low-PO 4 invertase yielded four components with p I values from 3.96 to 4.40, and yeast internal invertase (a mannan- and PO 4-free, cytoplasmic isozyme) was isoelectric at approx. pH 4.5. The high-PO 4 invertase was considerably more heterogeneous, with two major species of p I 3.65 and 3.32 and a highly acidic component of p I < 2.7; however, the mannose/PO 4 ratio of each species was approximately the same. PO 4-gradient elution from hydroxyapatite resolved the high-PO 4 invertase into five isozymes of increasing acidity and mannan content. Since the mannose/PO 4 ratios of these invertase species are constant, the increase in the mannan/protein (and, therefore PO 4/protein) ratio is apparently responsible for the microheterogeneity of phosphoinvertase.

Regulation of Isoenzymes and Secretion of Invertase in Bakers' Yeast

A method for estimating in intact yeast cells secreted and internal invertase activities was used for following hexose-repression and derepression of invertase secretion in the course of culture cycles on glucose medium. Derepression of secretion occurs abruptly when the glucose present in the medium is nearly exhausted. Time points of derepression and repression of invertase secretion can be determined by following the relative contents of the cultures in external and internal activities. Hexose-repression of invertase secretion could not be observed in spheroplasts. Changes of vinertase isoenzyme patterns upon derepression of secretion suggest that the small isoenzyme is not, or only partially involved in the synthesis of the secreted glycoprotein.

Properties of the internal invertase of yeast, Saccharomyces cerevisiae

1. 1. Internal yeast invertase (β- d-fructofuranoside fructohydrolase, EC 3.2.1.26) has been isolated and purified from fresh, sonicated Saccharomyces cerevisiae. The specific activity of the pure enzyme was 3128 μmoles sucrose hydrolyzed per ml per min per mg of protein at 25°. The enzyme has an elution volume 1.6 times the void volume on Sephadex G-200 at pH 7.5. 2. 2. A specific, reversible inhibitor effect of cations, notably NH 4 + was found. 3. 3. The internal yeast invertase is inhibited by iodine and the activity is regenerated by mercaptoethanol in an identical way to external invertase. The internal enzyme is also inhibited by cyanogen bromide in a two-step reaction. There is a fast, pH-dependent inhibition and a slow inhibition with a non-ionizing species; the rate of the latter reaction is affected slightly by pH. The slow reaction had a pseudo-first-order rate constant between 22 and 75·10 −6 sec −1 between pH 4.0 and pH 8.0, respectively. K 2PtCl 4 inhibits internal yeast invertase non-competitively with a dissociation constant of 0.025 M at pH 4.9. 4. 4. Internal iodine-invertase has a V which is 40% the value for the native enzyme; the K m of iodine invertase is 1.5 times the K m of the native enzyme. 5. 5. Internal invertase has transferring activity. Internal iodine-invertase has qualitatively the same activity. We were unable to show the formation of aniline fructosides with internal invertase, although we demonstrated such synthesis with external yeast invertase. External invertase can transfer fructose to methanol and to ethanol but not to glycollic acid. It can synthesize the O- fructoside with ethanol but not the analogous S- fructoside with ethanethiol. 6. 6. We conclude that the inhibition by iodine is due to the reversible oxidation of a reactive methionine sulphur atom. The slow inhibition by cyanogen bromide is also due to reaction at a methionine sulphur atom; but these are different methionine residues and, consequently, we suggest that there are two methionine residues at or near the active site of yeast invertase. 7. 7.The data presented are consistent with the mechanism of action proposed for external invertase by A. Waheed and S. Shall ( Biochim. Biophys. Acta, 242 (1971) 172) and we suggest that the external and internal yeast invertases have identical mechanisms of reaction.