Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.26 (invertase)
4,927 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Production of heterologous proteins by yeast secretion imposes additional factors that need to be considered, which do not appear with production by direct expression. These include additional intracellular polypeptide processing dynamics through the secretory organelles and the protein concentration in the culture medium, which is the usual final destination of the product. Optimal control theory is applied to optimize fed-batch production of secreted protein. We maximize an objective function that includes both total production rate and product concentration. A mutant invertase is chosen as the model heterologous secretory protein. Optimal control control strategies have been obtained for the use of two different promoters for the gene transcription, a dere-pressible SUC2 promoter and a strong glycolytic GPD promoter. With the use of the strong GPD promoter, achieving maximum production occurs on the singular arc of maximum specific growth rate. As the object switches to maximum product concentration, operation occurs for longer periods of time at a slow glucose singular arc condition. The optimal control for maximizing protein production with the weak SUC2 promoter requires transitions between high and low glucose concentrations associated with multiple distinct singular arc conditions. For maximum product concentration, the high concentration branches of the singular arc supporting maximum growth rate and maximum secretion rate disappear. Operation stays essentially on the low glucose concentration branch of the singular arc, which maximizes the protein production rate and minimizes the dilution of the broth product concentration.
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PMID:Effect of transcription promoters on the optimal production of secreted protein in fed-batch reactors. 136 71

The enzyme beta-lactamase, a secretory protein that is located in the Escherichia coli periplasmic space, can be highly expressed in Saccharomyces cerevisiae. Although the protein can cross eukaryotic membranes, it is only inefficiently secreted by yeast. To determine whether the lack of secretion in yeast is due to the nature of the bacterial signal sequence, it was replaced with the signal peptide of yeast invertase. The presence of the invertase signal peptide led to beta-lactamase secretion of up to 75%. The results indicate that the bacterial signal peptide is not functional in yeast, although cleavage can take place at the authentic processing site. The mature enzyme does not interfere with the yeast secretion pathway.
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PMID:Bacterial beta-lactamase is efficiently secreted in Saccharomyces cerevisiae under control of the invertase signal sequence. 152 52

Sequence comparison of the primary structure of the yeast Schwanniomyces occidentalis glucoamylase (GAM) with GAMs in different microorganisms did not reveal significant similarities. By contrast, striking similarities were, surprisingly, found with 3 mammalian secretory and integral membrane proteins: the 2 subunits of intestinal brush border sucrase-isomaltase and human lysosomal alpha-glucosidase. The similarities among these proteins are found as clusters of up to 8 amino acids and distributed all over the protein sequences. The major sequence differences are found in the N-terminal regions accounting, probably, for the different cellular locations of these proteins. The high level of similarities between sucrase, isomaltase, Sch. occidentalis GAM and human lysosomal alpha-glucosidase suggest that these proteins are derived from the same ancestral gene. To our knowledge, this is the first report that describes similarities between a yeast secretory protein and mammalian secretory and integral membrane proteins.
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PMID:Striking structural and functional similarities suggest that intestinal sucrase-isomaltase, human lysosomal alpha-glucosidase and Schwanniomyces occidentalis glucoamylase are derived from a common ancestral gene. 174 81

The Saccharomyces cerevisiae SEC14 gene encodes a cytosolic factor that is required for secretory protein movement from the Golgi complex. That some conservation of SEC14p function may exist was initially suggested by experiments that revealed immunoreactive polypeptides in cell extracts of the divergent yeasts Kluyveromyces lactis and Schizosaccharomyces pombe. We have cloned and characterized the K. lactis SEC14 gene (SEC14KL). Immunoprecipitation experiments indicated that SEC14KL encoded the K. lactis structural homolog of SEC14p. In agreement with those results, nucleotide sequence analysis of SEC14KL revealed a gene product of 301 residues (Mr, 34,615) and 77% identity to SEC14p. Moreover, a single ectopic copy of SEC14KL was sufficient to render S. cerevisiae sec14-1(Ts) mutants, or otherwise inviable sec14-129::HIS3 mutant strains, completely proficient for secretory pathway function by the criteria of growth, invertase secretion, and kinetics of vacuolar protein localization. This efficient complementation of sec14-129::HIS3 was observed to occur when the rates of SEC14pKL and SEC14p synthesis were reduced by a factor of 7 to 10 with respect to the wild-type rate of SEC14p synthesis. Taken together, these data provide evidence that the high level of structural conservation between SEC14p and SEC14pKL reflects a functional identity between these polypeptides as well. On the basis of the SEC14p and SEC14pKL primary sequence homology to the human retinaldehyde-binding protein, we suggest that the general function of these SEC14p species may be to regulate the delivery of a hydrophobic ligand to Golgi membranes so that biosynthetic secretory traffic can be supported.
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PMID:Cloning and characterization of Kluyveromyces lactis SEC14, a gene whose product stimulates Golgi secretory function in Saccharomyces cerevisiae. 219 63

SEC62 is required for the import of secretory protein precursors into the endoplasmic reticulum (ER) of Saccharomyces cerevisiae. The DNA sequence of SEC62 predicts a 32-kDa polypeptide with two potential membrane-spanning segments. Two antisera directed against different portions of the SEC62 coding region specifically detected a 30-kDa polypeptide in cell extracts. A combination of subcellular fractionation, detergent and alkali extraction, and indirect immunofluorescence studies indicated that Sec62p is intimately associated with the ER membrane. Protease digestion of intact microsomes and analysis of the oligosaccharide content of a set of Sec62p-invertase hybrid proteins suggested that Sec62p spans the ER membrane twice, displaying hydrophilic amino- and carboxy-terminal domains towards the cytosol. Sec62p-invertase hybrid proteins that lack the Sec62p C terminus failed to complement the sec62-l mutation and dramatically inhibited the growth of sec62-l cells at a normally permissive temperature. The inhibitory action of toxic Sec62p-invertase hybrids was partially counteracted by the overexpression of Sec63p. Taken together, these data suggest that the C-terminal domain of Sec62p performs an essential function and that the N-terminal domain associates with other components of the translocation machinery, including Sec63p.
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PMID:Structural and functional dissection of Sec62p, a membrane-bound component of the yeast endoplasmic reticulum protein import machinery. 223 30

We have recently shown that secretion of invertase is not inhibited in the yeast Saccharomyces cerevisiae during mitosis, but continues, as during interphase. This is in contrast with the mammalian cell, where membrane traffic stops at the onset of prometaphase. Here we extend our findings by showing that the bulk of the cell surface glycoproteins and mannans, as well as the yeast pheromone alpha-factor, traverse the secretory pathway during mitosis. We show that the mitotic cells are able to carry out several types of post-translational modification of secretory proteins. (a) The secretory protein invertase was oligomerized and extensively glycosylated, (b) the N-glycan cores of bulk-cell surface mannans were extended with outer chains, (c) some N-glycans were phosphorylated, (d) the protein-bound O-glycans were extended up to tetramannosides, (e) prepro-ka-factor was proteolytically processed to alpha-factor molecules. We conclude that the secretory pathway in yeast remains fully functional throughout the cell cycle.
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PMID:Post-translational modifications in mitotic yeast cells. 267 83

We have determined that prepro-carboxypeptidase Y and a truncated form of pre-invertase can be translocated across the yeast microsomal membrane post-translationally in a homologous in vitro system. The yeast secretory protein prepro-alpha-factor which was previously shown to be an efficient posttranslational translocation substrate is therefore not unique in this regard, but rather the yeast ER protein translocation machinery is generally capable of accepting substrates from a ribosome-free, soluble pool. However, within our detection limits, full-length pre-invertase could not be translocated posttranslationally, but was translocated co-translationally. This indicates that not every fully synthesized pre-protein can use this pathway, presumably because normal or aberrant folding characteristics can interfere with translocation competence.
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PMID:Prepro-carboxypeptidase Y and a truncated form of pre-invertase, but not full-length pre-invertase, can be posttranslationally translocated across microsomal vesicle membranes from Saccharomyces cerevisiae. 328 44

Yeast secretory mutant sec53 cells accumulate inactive secretory glycoprotein precursors that remain associated with the endoplasmic reticulum (ER) at the restrictive temperature (37 degrees C). The possibility that precursor polypeptides fail to penetrate completely into the ER lumen was tested by examining the protease accessibility of accumulated invertase, mating pheromone precursor prepro-alpha-factor and the vacuolar protein precursor procarboxypeptidase Y in cell lysates. In all three cases, the secretory protein precursors are protected from the action of exogenous protease unless the membrane is permeabilized by including Triton X-100 or saponin in the incubation. These results suggest that the sec53 defect allows complete polypeptide translocation. Consistent with this interpretation, the precursor of invertase accumulates in a signal peptide-processed form. In addition, invertase and prepro-alpha-factor precursors contain a small amount of possibly aberrant carbohydrate. In mutant cells or in wild type cells treated with tunicamycin, a 10-kDa fragment of the N terminus of mature invertase assumes a conformation that is resistant to trypsin with or without detergent. This domain may be associated with an ER protein or may simply assume an unusual conformation as a consequence of deficient glycosyl modification.
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PMID:Product of SEC53 is required for folding and glycosylation of secretory proteins in the lumen of the yeast endoplasmic reticulum. 329 55

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.
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PMID:Invertase beta-galactosidase hybrid proteins fail to be transported from the endoplasmic reticulum in Saccharomyces cerevisiae. 644 5

Emp24p is a type I transmembrane protein that is involved in secretory protein transport from the endoplasmic reticulum (ER) to the Golgi complex. A yeast mutant that lacks Emp24p (emp24 delta) is viable, but periplasmic invertase and the glycosylphosphatidyl-inositol-anchored plasma membrane protein Gas1p are delivered to the Golgi apparatus with reduced kinetics, whereas transport of alpha-factor, acid phosphatase and two vacuolar proteins is unaffected. Oligomerization and protease digestion studies of invertase suggest that the selective transport phenotype observed in the emp24 delta mutant is not due to a defect in protein folding or oligomerization. Consistent with a role in ER to Golgi transport, Emp24p is a component of COPII-coated, ER-derived transport vesicles that are isolated from a reconstituted in vitro budding reaction. We propose that Emp24p is involved in the sorting and/or concentration of a subset of secretory proteins into ER-derived transport vesicles.
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PMID:The absence of Emp24p, a component of ER-derived COPII-coated vesicles, causes a defect in transport of selected proteins to the Golgi. 772 11


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