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)

We have used cell fusion to address the question of whether macromolecules are rapidly exchanged between lysosomes. Donor cell lysosomes were labeled by the long-term internalization of the fluid-phase pinocytic markers, invertase (sucrase), Lucifer Yellow, FITC-conjugated dextran, or Texas red-conjugated dextran. Recipient cells contained lysosomes swollen by long-term internalization of dilute sucrose or marked by an overnight FITC-dextran uptake. Cells were incubated for 1 or 2 h in marker-free media before cell fusion to clear any marker from an endosomal compartment. Recipient cells were infected with vesicular stomatitis virus as a fusogen. Donor and recipient cells were co-cultured for 1 or 2 h and then fused by a brief exposure to pH 5. In all cases, extensive exchange of content between donor and recipient cell lysosomes was observed at 37 degrees C. Incubation of cell syncytia at 17 degrees C blocked lysosome/lysosome exchange, although a "priming" process(es) appeared to occur at 17 degrees C. The kinetics of lysosome/lysosome exchange in fusions between cells containing invertase-positive lysosomes and sucrose-positive lysosomes indicated that lysosome/lysosome exchange was as rapid, if not more rapid, than endosome/lysosome exchange. These experiments suggest that in vivo the lysosome is a rapidly intermixing organellar compartment.
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PMID:Chinese hamster ovary cell lysosomes rapidly exchange contents. 244 96

Proteins destined for the nucleus contain nuclear localization sequences, short stretches of amino acids responsible for targeting them to the nucleus. We show that the first 29 amino acids of GAL4, a yeast DNA-binding protein, function efficiently as a nuclear localization sequence when fused to normally cytoplasmic invertase, but not when fused to Escherichia coli beta-galactosidase. Moreover, the nuclear localization sequence from simian virus 40 T antigen functions better when fused to invertase than when fused to beta-galactosidase. A single amino acid change in the T-antigen nuclear localization sequence inhibits the nuclear localization of simian virus 40-invertase and simian virus 40-beta-galactosidase in Saccharomyces cerevisiae. From these results, we conclude that the relative ability of a nuclear localization sequence to act depends on the protein to which it is linked.
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PMID:Context affects nuclear protein localization in Saccharomyces cerevisiae. 249

The construction of two fused genes is described. One involves the in-frame fusion of the yeast prepro-alpha-factor coding sequence, and the Escherichia coli lac Z gene. The second gene fusion utilizes a 103 bp yeast invertase NH2-terminal coding sequence at the fusion junction of the hybrid gene described above. The gene fusions, under the control of the alpha-factor promoter, expressed active beta-galactosidase in alpha haploid yeast cells. The activity could be regulated in a temperature-sensitive sir3 mutant. The incorporation of the invertase coding sequence at the MF alpha 1-lacZ fusion junction provided significantly higher levels of beta-galactosidase activity. A substantial quantity of the hybrid proteins generated from the gene fusions was primarily localized in the intracellular membranes of yeast cells, while a processed form could be secreted into the periplasm.
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PMID:Alpha-factor leader sequence-directed transport of Escherichia coli beta-galactosidase in the secretory pathway of Saccharomyces cerevisiae. 250 25

When MATa cells of Saccharomyces cerevisiae have been treated with the mating hormone alpha-factor an increase in chitin synthase zymogen, as well as chitin content in the cell-wall fraction, have been reported. With a DNA probe derived from the cloned CHS1 gene that codes for chitin synthase I [Bulawa, C. E., Slater, M., Cabib, E., Au-Young, J., Sburlati, A., Adair, W. L. and Robbins, P. (1986) Cell 46, 213-225] a Northern analysis was conducted of CHS1-specific transcripts. alpha-Factor-treated MATa cells revealed more than sixfold elevated steady-state levels of CHS1 mRNA as compared to control cells. MAT alpha cells responded the same way when treated with a-factor although induction rate was somewhat smaller. After hormone application a rapid increase in CHS1 mRNA levels could be observed that occurred also in the absence of ongoing protein synthesis. In order to minimize possible side effects of CHS1-coding sequences on expression and mRNA stability a CHS1::SUC2 chimaeric gene was constructed where 730 bp of the CHS1 promoter region (+20 bp of the coding region) were fused in frame to a fragment of the SUC2 coding region. The fusion protein exhibits invertase activity that has been used to monitor CHS1 promoter activity. By analysis of shortened versions of the CHS1 promoter a 94-bp DNA fragment has been identified that confers hormone inducibility to the CHS1 promoter. According to the published sequence of the CHS1 gene, this fragment contains four repeats of a TGAAACA consensus sequence previously identified in the alpha-factor-inducible BAR1 promoter [Kronstad, J. W., Holly, J. A. and MacKay, V. L. (1987) Cell 50, 369-377]. This heptamer may represent the cis-acting element involved in mating-hormone-mediated gene expression in yeast.
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PMID:Hormone-induced expression of the CHS1 gene from Saccharomyces cerevisiae. 252

When nuclear localization sequences (termed NLS) are placed at the N terminus of cytochrome c1, a mitochondrial inner membrane protein, the resulting hybrid proteins do not assemble into mitochondria when synthesized in the yeast Saccharomyces cerevisiae. Cells lacking mitochondrial cytochrome c1, but expressing the hybrid NLS-cytochrome c1 proteins, are unable to grow on glycerol since the hybrid proteins are associated primarily with the nucleus. A similar hybrid protein with a mutant NLS is transported to and assembled into the mitochondria. To identify proteins that might be involved in recognition of nuclear localization signals, we isolated conditional-lethal mutants (npl, for nuclear protein localization) that missorted NLS-cytochrome c1 to the mitochondria, allowing growth on glycerol. The gene corresponding to one complementation group (NPL1) encodes a protein with homology to DnaJ, an Escherichia coli heat shock protein. npl1-1 is allelic to sec63, a gene that affects transit of nascent secretory proteins across the endoplasmic reticulum. Rothblatt, J. A., R. J. Deshaies, S. L. Sanders, G. Daum, and R. Schekman. 1989. J. Cell Biol. 109:2641-2652. The npl1 mutants reported here also weakly affect translocation of preprocarboxypeptidaseY across the ER membrane. A normally nuclear hybrid protein containing a NLS fused to invertase and a nucleolar protein are not localized to the nucleus in npl1/sec63 cells at the nonpermissive temperature. Thus, NPL1/SEC63 may act at a very early common step in localization of proteins to the nucleus and the ER. Alternatively, by affecting ER and nuclear envelope assembly, npl1 may indirectly alter assembly of proteins into the nucleus.
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PMID:A yeast gene important for protein assembly into the endoplasmic reticulum and the nucleus has homology to DnaJ, an Escherichia coli heat shock protein. 255 4

DNA segments encoding signal peptides from mouse alpha-amylase, yeast acid phosphatase, and yeast invertase were fused in frame to a barley (1-3,1-4)-beta-glucanase cDNA gene and expressed in yeast cells under the control of the phosphoglycerate kinase gene promoter. Pure beta-glucanase is obtained by gel filtration of concentrated yeast cell supernatant. It was shown that the glucanase pre-protein was specifically processed and the mature protein efficiently secreted when the yeast invertase signal sequence directed secretion.
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PMID:Processing and secretion of barley (1-3,1-4)-beta-glucanase in yeast. 267 77

Synthetic oligonucleotides coding for the yeast invertase secretion signal peptide were fused to the gene for the mature form of human interferon (huIFN-alpha 2). Two plasmids (E3 and F2) were constructed. E3 contained the invertase signal codons in a reading frame with the mature huIFN-alpha 2 gene. F2 had a deletion of the codon for alanine at amino acid residue-5 in the invertase signal and an addition of a methionine codon located between the coding sequences for the invertase signal and mature huIFN-alpha 2. Both hybrid genes were located adjacent to the promoter from the 3-phosphoglycerate kinase gene on the multicopy yeast expression plasmid, YEp1PT. Yeast transformants containing these plasmids produced somewhat more IFN than did the same expression plasmid containing the IFN gene with its human secretion signal sequence. HuIFN-alpha 2, purified from the medium of yeast cells containing E3, was found to be processed at the correct site. The huIFN-alpha 2 made by plasmid F2 was found to be completely processed at the junction between the invertase signal (a variant) and the methionine of methionine-huIFN-alpha 2. These results strongly suggested that the invertase signal (or its variant) attached to huIFN was efficiently recognized by the presumed signal recognition particle and was cleaved by the signal peptidase in the yeast cells. These results also suggested that amino acid changes on the right side of the cleavage site did not necessarily prevent cleavage or secretion.
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PMID:Saccharomyces cerevisiae secretes and correctly processes human interferon hybrid proteins containing yeast invertase signal peptides. 302 6

We have mapped a sequence determinant in the vacuolar glycoprotein carboxypeptidase Y (CPY) that directs intracellular sorting of this enzyme. Through the study of hybrid proteins, consisting of amino-terminal segments of CPY fused to the secretory enzyme invertase, we have found that the N-terminal 50 amino acids of CPY are sufficient to direct delivery of a CPY-Inv hybrid protein to the yeast vacuole. Our data suggest that this 50 amino acid segment of CPY contains two distinct functional domains; an N-terminal signal peptide followed by a segment of 30 amino acids that contains the vacuolar sorting signal. Deletion of this putative vacuole sorting signal from an otherwise wild-type CPY protein leads to missorting of CPY. Furthermore, examination of the Asn-linked oligosaccharides present on CPY and CPY-Inv hybrid proteins suggests that an additional determinant in CPY specifies the extent to which these proteins are glycosylated in the Golgi complex.
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PMID:Distinct sequence determinants direct intracellular sorting and modification of a yeast vacuolar protease. 302 48

Various gene fusions between the arginine permease and invertase have been constructed in order to obtain information about whether parts of the CAN1 gene product can induce secretion of biologically active invertase missing its own signal sequence. A construction containing 30 N-terminal amino acid residues of the CAN1 gene product fused to invertase was not secreted. When the CAN1 portion was elongated to 477 or 560 amino acid residues, secretion of the fusion proteins was observed. A fusion lacking 59 amino acids at the amino-terminal end of the arginine permease was also secreted. These results indicate that the amino-terminal end of the arginine permease is neither sufficient nor essential for membrane insertion; instead this enzyme should contain an internal targeting sequence facilitating secretion. Some general implications on the biosynthesis and topology of membrane proteins are also discussed as well as the homology with histidine permease.
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PMID:CAN1-SUC2 gene fusion studies in Saccharomyces cerevisiae. 332 76

Signal sequences of Saccharomyces cerevisiae invertase and alpha-factor pheromone were tested for the ability to mediate protein transport through the inner membrane of Escherichia coli by fusion to bacterial beta-lactamase lacking the signal sequence (blaS0). Both types of transformants exhibited ampicillin resistance in accordance with the transport of the fused protein to the periplasmic compartment. This compartment contained most of the beta-lactamase activity present in the cell. Therefore, the tested yeast signal sequences, which conferred translocation of their proteins across the membrane of the endoplasmic reticulum in S. cerevisiae, can provide the same function in E. coli. The screening for ampicillin resistance among blaS0 fusions provides a convenient method for the isolation of functional yeast and possibly higher eucaryotic signal sequences.
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PMID:Mediation, by Saccharomyces cerevisiae translocation signals, of beta-lactamase transport through the Escherichia coli inner membrane and sensitive method for detection of signal sequences. 353 Nov 85


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