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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)

We have optimized the codons in an immunoglobulin kappa chain gene to those preferred in the yeast Saccharomyces cerevisiae. The mutant and wild type kappa chain genes were each fused with a synthetic invertase signal peptide that also contained only yeast-preferred codons, and expressed in the F762 yeast strain. The use of yeast-preferred codons resulted in a more than 5-fold increase in the rate of synthesis and at least a 50-fold increase in the steady state level of protein.
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PMID:Evaluation of foreign gene codon optimization in yeast: expression of a mouse IG kappa chain. 136 71

To study the sorting of proteins in Dictyostelium discoideum, we used vector constructs that contain cDNA coding for the entire beta-hexosaminidase protein to prepare transformants of a mutant that lacks this enzyme activity. These transformants overexpressed active, normally processed beta-hexosaminidase. The overexpressed enzyme colocalized with other acid hydrolases in the soluble fraction of vesicles in the lysosomal region of Percoll gradients. The sorting of other hydrolases was unaltered. We also prepared transformants with constructs that contain 22 (Hex 22-Inv), 70 (Hex 70-Inv), and 532 (Hex 532-Inv) amino-terminal amino acids from beta-hexosaminidase fused in frame with the coding sequence for the yeast SUC2 gene product, invertase. Fusion molecular masses were those expected for fully N-glycosylated proteins. Hex 22-Inv was rapidly (t1/2 less than 30 min) and quantitatively secreted. The others were slowly (t1/2 greater than 5 h) and partially secreted. Each expressed invertase activity. During growth, the invertase activity of Hex 70-Inv and Hex 532-Inv was retained to the same extent as that of endogenous lysosomal enzymes. Most of the Hex 70-Inv migrated in Percoll gradients with vesicles of intermediate density (d = 1.055), but a portion co-migrated with lysosomal enzymes at d = 1.08. Hex 70-Inv was sulfated, and its N-glycosides were resistant to endoglycosidase H, indicating Golgi processing. Hex 70-Inv and Hex 532-Inv, like endogenous lysosomal enzymes, were subject to developmentally induced secretion.
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PMID:A sequence in beta-hexosaminidase from Dictyostelium discoideum required for sorting of proteins to a compartment involved in developmentally induced secretion. 153 76

We have analyzed a series of Moloney murine leukemia (M-MuLV) envelope (env) protein fusions to the marker proteins invertase and placental alkaline phosphatase (PLAP), expressed in Psi2 retrovirus packaging cells. The yeast invertase protein, fused at its third amino acid residue to the amino-terminal signal sequence and 17 residues of the mature M-MuLV env protein, retained its enzymatic activity and was secreted from mammalian cells. However, env protein fusions to the C-terminal portion of invertase were inactive. In contrast, some, but not all, env protein fusions at the C-terminal region of PLAP were enzymatically active: PLAP fusions containing long C-terminal portions of env localized to the rough endoplasmic reticulum (RER) and possessed low enzyme activity levels, while fusion constructs containing relatively short portions of the M-MuLV env gene localized to the Golgi and had higher activity levels. Those proteins that localized to the Golgi also were processed, in part, to forms of 67 to 68 kDa, the size of the mature PLAP protein. Since PLAP ordinarily is transferred to a phosphatidyl-inositol glycan tail (PIG-tail) in the Golgi and then transported to the plasma membrane, it appears that Golgi-localized PLAP-env fusions are processed imperfectly. PLAP itself, when expressed in Psi2 cells, accumulated at the plasma membrane and, unlike the PIG-tailed Thy-1 protein, was not incorporated into virus particles. Thus, the reported incorporation of the Thy-1 protein into M-MuLV virions does not appear to be a consequence of its glycoprotein tail.
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PMID:Retroviral envelope protein fusions to secreted and membrane markers. 158 54

Legumin subunits are synthesized as precursor polypeptides and are transported into protein storage vacuoles in field bean cotyledons. We expressed a legumin subunit in yeast and found that in these cells it is also transported into the vacuoles. To elucidate vacuolar targeting information, we constructed gene fusions of different legumin propolypeptide segments with either yeast invertase or chloramphenicol acetyltransferase as reporters for analysis in yeast or plant cells, respectively. In yeast, increasing the length of the amino-terminal segment increased the portion of invertase directed to the vacuole. Only the complete legumin alpha chain (281 amino acids) directed over 90% to the vacuole. A short carboxy-terminal legumin segment (76 amino acids) fused to the carboxy terminus of invertase also efficiently targeted this fusion product to yeast vacuoles. With amino-terminal legumin-chloramphenicol acetyltransferase fusions expressed in tobacco seeds, efficient vacuolar targeting was obtained only with the complete alpha chain. We conclude that legumin contains multiple targeting information, probably formed by higher structures of relatively long peptide sequences.
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PMID:Different legumin protein domains act as vacuolar targeting signals. 184 24

We have fused a cDNA gene encoding mature human serum albumin (HSA) to several secretory leader-encoding sequences. The hybrid genes were cloned into an episomal vector under the control of several yeast promoters and then introduced into yeast cells. The GAL1 promoter in combination with either the native HSA pre-sequence or a modified HSA pre-sequence gave the highest production of immunoreactive HSA, 90 mg/liter being reached in a shake flask culture. The invertase pre-sequence, the mating factor alpha 1 prepro-sequence, and the modified HSA pre-sequence directed accurate processing. In contrast, the chicken lysozyme pre-sequence and the native HSA pre-sequence directed incorrect processing. Episomal vectors were unstable within the host cells under non-selective culture conditions. To improve the plasmid stability, the hybrid genes were incorporated into an integrative vector. Transformants carrying multicopies of the plasmid integrated at the LEU2 locus stably secreted HSA. The highest yield of 65 mg/liter in a shake flask culture was obtained with the combination of the yeast glyceraldehyde-3-phosphate dehydrogenase promoter and the modified HSA pre-sequence. By constructing transformed strains containing multicopies of plasmids integrated at both the chromosome LEU2 and HIS4 loci, we have obtained a stable strain that continuously secretes as much as 85 mg HSA per liter of culture medium.
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PMID:Secretory expression of the human serum albumin gene in the yeast, Saccharomyces cerevisiae. 193 15

The mechanism of yolk deposition into developing oocytes of Drosophila was investigated by following the fate of a reporter protein fused to a vitellogenin, or yolk polypeptide (YP). Embryos were transformed with a hybrid gene consisting of the promotor and amino terminal 430 codons of the Yp2 gene fused to the cytoplasmic form of the invertase gene from the yeast Saccharomyces cerevisiae. RNA hybridization experiments with established lines of transformed flies showed that the hybrid gene was expressed in female fat bodies and ovaries but not in any male cells. Immunoblotting and endoglycosidase digestion showed that the hybrid protein was secreted from fat body cells via the secretory pathway, transported in hemolymph, and sequestered into developing oocytes. Transfusion experiments with hemolymph and pure invertase showed that sequestration of invertase depended on its attachment to YP. Immunocytochemistry demonstrated that the hybrid protein became localized in yolk granules as oocytes developed. Females homozygous for the fusion gene are generally sterile; their eggs containing the hybrid protein often collapse and their embryos fail to develop, suggesting that the structure of the yolk polypeptides is important for embryonic development. These experiments show that YP2 carries structural information sufficient to direct a reporter protein from fat body cells, through the hemolymph, and into the yolk granules of developing oocytes. This work provides a means of identifying the features of yolk polypeptides that are responsible for their deposition into yolk during oogenesis.
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PMID:Vitellogenesis in Drosophila: sequestration of a yolk polypeptide/invertase fusion protein into developing oocytes. 211 78

Phytohemagglutinin (PHA), the seed lectin of the common bean, accumulates in protein storage vacuoles of storage parenchyma cells in cotyledons. When expressed in yeast, PHA is efficiently targeted to the yeast vacuole [Tague and Chrispeels (1987). J. Cell Biol. 105, 1971-1979]. To identify vacuolar sorting information in PHA, a series of 3' deletions of the PHA gene were fused in-frame to a truncated yeast invertase gene. An amino-terminal portion of PHA composed of a 20-residue signal sequence and 43 residues of the mature protein efficiently targeted invertase to the yeast vacuole. Internal deletions in a short PHA-invertase fusion showed that targeting information exists between residues 14 and 23 of mature PHA. Based on examinations of three-dimensional structures of related lectins, only a portion of these residues would be available on the surface of PHA for interaction with a putative receptor. Amino acid replacements at these positions in a PHA-invertase hybrid caused secretion of the invertase. The results indicate the presence of a vacuolar targeting domain in PHA that is centered at position 19 of the mature protein. This sequence of PHA also shows sequence identity to a vacuolar sorting domain characterized in yeast carboxypeptidase Y. Single amino acid alterations in a short PHA-invertase hybrid protein that caused the highest levels of secretion introduced a glycosylation site at position 21 of PHA. This observation suggests that glycan addition may interfere with recognition of a sorting determinant. These same amino acid changes did not dramatically increase secretion in a long PHA-invertase fusion or in PHA itself. Thus, a second domain of PHA may function in concert with the first one to bring about correct targeting of PHA.
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PMID:A short domain of the plant vacuolar protein phytohemagglutinin targets invertase to the yeast vacuole. 215 75

Three randomly derived sequences that can substitute for the signal peptide of Saccharomyces cerevisiae invertase were tested for the efficiency with which they can translocate invertase or beta-galactosidase into the endoplasmic reticulum. The rate of translocation, as measured by glycosylation, was estimated in pulse-chase experiments to be less than 6 min. When fused to beta-galactosidase, these peptides, like the normal invertase signal sequence, direct the hybrid protein to a perinuclear region, consistent with localization to the endoplasmic reticulum. The diversity of function of random peptides was studied further by immunofluorescence localization of proteins fused to 28 random sequences: 4 directed the hybrid to the endoplasmic reticulum, 3 directed it to the mitochondria, and 1 directed it to the nucleus.
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PMID:Efficiency and diversity of protein localization by random signal sequences. 216 May 95

A plasmid-encoded gene for a hybrid pre-protein containing most of the bovine prolactin signal peptide (SpPRL) fused to the mature sequence of yeast invertase (IVT) was expressed and the product was processed and secreted by yeast. However, the level of IVT activity was reduced about six-fold when compared to that obtained with the wild type (wt) invertase signal peptide (SpIVT). When the 5'-untranslated sequence of the hybrid mRNA was truncated by 29 nucleotides, a 2.5-fold increase in secreted IVT was observed. Replacement of the PRL codons with preferred yeast codons did not result in any improvement in the production of secreted IVT. An increase in IVT activity to the level observed with the wt SpIVT was obtained by replacement of the Gly residue located between the N terminus and the central lipophilic region of the SpPRL by Ala. Since this amino acid replacement results in a higher probability of the SpPRL assuming an alpha-helical conformation, it suggests that the secondary structure of this region is important in recognition by the yeast secretory apparatus.
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PMID:Changes in a mammalian signal sequence required for efficient protein secretion by yeasts. 218 92

Chimeric genes consisting of the coding sequence of the yeast invertase gene suc 2 and different N-terminal portions of the potato-derived vacuolar protein proteinase inhibitor II fused to the 35S CaMV promoter and the poly-A site of the octopine synthase gene were transferred into tobacco and Arabidopsis thaliana plants using Agrobacterium based systems. Regenerated transgenic plants display a 50- to 500-fold higher invertase activity compared to non-transformed control plants. This invertase is N-glycosylated and efficiently secreted from the plant cell leading to its apoplastic location. Whereas expression of the invertase does not lead to drastic changes in transgenic Arabidopsis thaliana plants, transgenic tobacco plants show dramatic changes with respect to development and phenotype. Expression of the invertase leads to stunted growth due to reduction of internodal distances, to development of bleached and/or necrotic regions in older leaves and to suppressed root formation. In mature leaves, high levels of soluble sugars and starch accumulate. These carbohydrates do not show a diurnal turnover. The accumulation of carbohydrate is accompanied by an inhibition of photosynthesis, and in tobacco, by an increase in the rate of respiration. Measurements in bleached versus green areas of the same leaf show that the bleached section contains high levels of carbohydrates and has lower photosynthesis and higher respiration than green sections. It is concluded that expression of invertase in the cell wall interrupts export and leads to an accumulation of carbohydrates and inhibition of photosynthesis.
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PMID:Expression of a yeast-derived invertase in the cell wall of tobacco and Arabidopsis plants leads to accumulation of carbohydrate and inhibition of photosynthesis and strongly influences growth and phenotype of transgenic tobacco plants. 220 36


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