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)

When the extracellular domain of rat low-affinity nerve growth factor receptor (NGFRe) was synthesized in Saccharomyces cerevisiae with the signal peptide of invertase, NGFRe was translocated to the endoplasmic reticulum (ER) and retained there. However, when NGFRe was fused to the C-terminus of the hsp150 delta-carrier, the hsp150 delta-NGFRe fusion protein was efficiently secreted to the growth medium with no apparent retention in the ER. The NGFRe portion was disulphide-bonded and its single N-glycosylation site was occupied. The hsp150 delta-carrier is an N-terminal signal peptide-containing fragment of a yeast secretory glycoprotein. Hsp150 delta-NGFRe, harvested from the culture medium, inhibited the cross-linking of [125I]NGF to authentic NGFR on the surface of human melanoma cells. Moreover, [125I]NGF could be chemically cross-linked to secretory hsp150 delta-NGFRe, suggesting that the NGFRe portion had adopted a ligand-binding conformation. However, inhibition of the cross-linking by unlabelled NGF was less effective than in the case of the authentic receptor. The hsp150 delta-carrier may have potential in the production of mammalian proteins, which require elaborate folding and disulphide formation in the ER.
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PMID:The hsp150 delta-carrier confers secretion competence to the rat nerve growth factor receptor ectodomain in Saccharomyces cerevisiae. 874 Apr 19

The assembly of functional proteins from fragments in vivo has been recently described for several proteins, including the secreted maltose binding protein in Escherichia coli. Here we demonstrate for the first time that split gene products can function within the eukaryotic secretory system. Saccharomyces cerevisiae strains able to use sucrose produce the enzyme invertase, which is targeted by a signal peptide to the central secretory pathway and the periplasmic space. Using this enzyme as a model we find the following: (i) Polypeptide fragments of invertase, each containing a signal peptide, are independently translocated into the endoplasmic reticulum (ER) are modified by glycosylation, and travel the entire secretory pathway reaching the yeast periplasm. (ii) Simultaneous expression of independently translated and translocated overlapping fragments of invertase leads to the formation of an enzymatically active complex, whereas individually expressed fragments exhibit no activity. (iii) An active invertase complex is assembled in the ER, is targeted to the yeast periplasm, and is biologically functional, as judged by its ability to facilitate growth on sucrose as a single carbon source. These observation are discussed in relation to protein folding and assembly in the ER and to the trafficking of proteins through the secretory pathway.
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PMID:Split invertase polypeptides form functional complexes in the yeast periplasm in vivo. 879 Mar 78

The human Fc fragments of immunoglobulin E (IgE-Fc) expressed in the mammalian COS cells, those mainly consisting of C epsilon 3-C epsilon 4 chains with or without Cys-328 known to be responsible for interchain disulfide bonding, were secreted into the culture medium directed by the yeast Saccharomyces cerevisiae invertase (SUC2) signal sequence (SUC2/Ig2 or SUC2/Ig, respectively) as well as by the human interleukin 2 receptor alpha chain signal sequence (IL2R/Ig2 or IL2R/Ig, respectively). For the binding activity to the human soluble alpha chain of high affinity receptor for IgE, IgE-Fc with Cys328, SUC2/Ig2 and IL2R/Ig2, were active but had smaller activity than native IgE. By comparison IgE-Fc without Cys328, SUC2/Ig and IL2R/Ig, showed lower activity than SUC2/Ig2 and IL2R/Ig2. Immunoprecipitation analysis revealed both SUC2/Ig and SUC2/Ig2 had molecular weights (M(r)s) and degrees of glycosylation similar to IL2R/Ig and IL2R/Ig2, respectively. These results suggested that in mammalian cells the SUC2 signal sequence was functional in directing the heterologous multimeric proteins to the endoplasmic reticulum, resulting in secretion of the active proteins. This finding might show merits on heterologous protein secretion systems.
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PMID:Secretion of active Fc fragments of immunoglobulin E directed by the yeast invertase signal sequence in mammalian cells. 913 Mar 68

Overproduction of an endoplasmic reticulum (ER)-resident membrane protein (cytochrome P450 52A3) and of a secretory protein (invertase) was used to study the regulation of the luminal ER protein Kar2p under conditions that lead to ER proliferation and secretory overload, respectively. In both cases we found (i) a significant increase of Kar2 protein and mRNA levels, (ii) a transcriptional regulation based on the the function of the 22 bp unfolded-protein-response element of the KAR2 promoter and (iii) an essential role of the transmembrane kinase Ire1p for upregulation of KAR2 gene expression. These results show that the same mechanism operates when KAR2 induction is triggered by overproduction of cytochrome P450 or invertase and that this mechanism shares the known features of the unfolded-protein-response pathway. Disruption of the IRE1 gene resulted in a marked decrease of the invertase protein levels produced. In contrast, a functional IRE1 gene was not required to reach high-level production of the integral membrane protein cytochrome P450 52A3, Moreover, IRE1 gene disruption did not prevent P450-induced ER proliferation. We suggest that Ire1p-mediated KAR2 induction is, in the case of cytochrome P450 52A3 overproduction, a process which follows on ER proliferation, thereby monitoring the increase of ER size and adjusting the level of Kar2p accordingly.
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PMID:Inducible membranes in yeast: relation to the unfolded-protein-response pathway. 936 46

We have cloned PEX15 which is required for peroxisome biogenesis in Saccharomyces cerevisiae. pex15Delta cells are characterized by the cytosolic accumulation of peroxisomal matrix proteins containing a PTS1 or PTS2 import signal, whereas peroxisomal membrane proteins are present in peroxisomal remnants. PEX15 encodes a phosphorylated, integral peroxisomal membrane protein (Pex15p). Using multiple in vivo methods to determine the topology, Pex15p was found to be a tail-anchored type II (Ncyt-Clumen) peroxisomal membrane protein with a single transmembrane domain near its carboxy-terminus. Overexpression of Pex15p resulted in impaired peroxisome assembly, and caused profound proliferation of the endoplasmic reticulum (ER) membrane. The lumenal carboxy-terminal tail of Pex15p protrudes into the lumen of these ER membranes, as demonstrated by its O-glycosylation. Accumulation in the ER was also observed at an endogenous expression level when Pex15p was fused to the N-terminus of mature invertase. This resulted in core N-glycosylation of the hybrid protein. The lumenal C-terminal tail of Pex15p is essential for targeting to the peroxisomal membrane. Furthermore, the peroxisomal membrane targeting signal of Pex15p overlaps with an ER targeting signal on this protein. These results indicate that Pex15p may be targeted to peroxisomes via the ER, or to both organelles.
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PMID:Overexpression of Pex15p, a phosphorylated peroxisomal integral membrane protein required for peroxisome assembly in S.cerevisiae, causes proliferation of the endoplasmic reticulum membrane. 940 62

There is growing evidence that yeast contains two efficient pathways of protein translocation across the endoplasmic reticulum membrane, one dependent on the signal-recognition particle (SRP) and the other independent. Their specificity, however, is largely obscure. For higher eukaryotes it has been shown that a high average hydrophobicity of the core region with a minimal length around six or seven amino acids, as well as a stabilized alpha-helix, are decisive structural features for translocation. Using yeast invertase as a secretory model protein, we have found that mutated signal sequences with Pro or Gly in the core, or having only four hydrophobic amino acids, are not functional in translocation across microsomal membranes of dog pancreas because they do not interact with the SRP. Expression of these mutant variants in Saccharomyces cerevisiae revealed that they are sorted independently of the SRP since translocation was not impaired in an SRP-deficient yeast strain. In contrast to this, wild-type invertase is translocated SRP-dependently in wild-type cells and shows a decreased translocation in SRP-deficient cells. By overexpression of Srp54p, but not of Hsc70p, the translocation defect of wild-type invertase in an SRP54 disruptant is restored. The data indicate that targeting of proteins to the endoplasmic reticulum in Saccharomyces cerevisiae seems to be more flexible than in higher eukaryotes as far as the structural requirements of signal sequences are concerned, and that the route taken is specified by the sequence.
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PMID:Sorting of invertase signal peptide mutants in yeast dependent and independent on the signal-recognition particle. 952 7

Mechanisms to acquire tolerance against heat, an important environmental stress condition, have evolved in all organisms, but are largely unknown. When Saccharomyces cerevisiae cells are pre-conditioned at 37 degrees C, they survive an otherwise lethal exposure to 48-50 degrees C, and form colonies at 24 degrees C. We show here that incubation of yeast cells at 48-50 degrees C, after pre-conditioning at 37 degrees C, resulted in inactivation of exocytosis, and in conformational damage and loss of transport competence of proteins residing in the endoplasmic reticulum (ER). Soon after return of the cells to 24 degrees C, membrane traffic was resumed, but cell wall invertase, vacuolar carboxypeptidase Y and a secretory beta-lactamase fusion protein remained in the ER for different times. Thereafter their transport competence was resumed very slowly with widely varying kinetics. While the proteins were undergoing conformational repair in the ER, their native counterparts, synthesized after shift of the cells to 24 degrees C, folded normally, by-passed the heat-affected copies and exited rapidly the ER. The Hsp70 homolog Lhs1p was required for acquisition of secretion competence of heat-damaged proteins. ER retention and refolding of heat-denatured glycoproteins appear to be part of the cellular stress response.
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PMID:Transient ER retention as stress response: conformational repair of heat-damaged proteins to secretion-competent structures. 958 May 65

The regulation of vesicular transport in eukaryotic cells involves Ras-like GTPases of the Ypt/Rab family. Studies in yeast and mammalian cells indicate that individual family members act in vesicle docking/fusion to specific target membranes. Using the two-hybrid system, we have now identified a 248 amino acid, integral membrane protein, termed Yip1, that specifically binds to the transport GTPases Ypt1p and Ypt31p. Evidence for physical interaction of these GTPases with Yip1p was also demonstrated by affinity chromatography and/or co-immunoprecipitation. Like the two GTPases, Yip1p is essential for yeast cell viability and, according to subcellular fractionation and indirect immunofluorescence, is located to Golgi membranes at steady state. Mutant cells depleted of Yip1p and conditionally lethal yip1 mutants at the non-permissive temperature massively accumulate endoplasmic reticulum membranes and display aberrations in protein secretion and glycosylation of secreted invertase. The results suggests for a role for Yip1p in recruiting the two GTPases to Golgi target membranes in preparation for fusion.
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PMID:Specific binding to a novel and essential Golgi membrane protein (Yip1p) functionally links the transport GTPases Ypt1p and Ypt31p. 972 32

Secretory proteins in eukaryotic cells are transported to the cell surface via the endoplasmic reticulum (ER) and the Golgi apparatus by membrane-bounded vesicles. We screened a collection of temperature-sensitive mutants of Saccharomyces cerevisiae for defects in ER-to-Golgi transport. Two of the genes identified in this screen were PRP2, which encodes a known pre-mRNA splicing factor, and RSE1, a novel gene that we show to be important for pre-mRNA splicing. Both prp2-13 and rse1-1 mutants accumulate the ER forms of invertase and the vacuolar protease CPY at restrictive temperature. The secretion defect in each mutant can be suppressed by increasing the amount of SAR1, which encodes a small GTPase essential for COPII vesicle formation from the ER, or by deleting the intron from the SAR1 gene. These data indicate that a failure to splice SAR1 pre-mRNA is the specific cause of the secretion defects in prp2-13 and rse1-1. Moreover, these data imply that Sar1p is a limiting component of the ER-to-Golgi transport machinery and suggest a way that secretory pathway function might be coordinated with the amount of gene expression in a cell.
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PMID:A link between secretion and pre-mRNA processing defects in Saccharomyces cerevisiae and the identification of a novel splicing gene, RSE1. 981

To examine the possibility of active recycling of Emp24p between the endoplasmic reticulum (ER) and the Golgi, we sought to identify transport signal(s) in the carboxyl-terminal region of Emp24p. Reporter molecules were constructed by replacing parts of a control invertase-Wbp1p chimera with those of Emp24p, and their transport rates were assessed. The transport of the reporter was found to be accelerated by the presence of the cytoplasmic domain of Emp24p. Mutational analyses revealed that the two carboxyl-terminal residues, leucine and valine (LV), were necessary and sufficient to accelerate the transport. The acceleration was sequence specific, and the terminal valine appeared to be more important. The LV residues accelerated not only the overall transport to the vacuole but also the ER to cis-Golgi transport, suggesting its function in the ER export. Hence the LV residues are a novel anterograde transport signal. The double-phenylalanine residues did not affect the transport by itself but attenuated the effect of the anterograde transport signal. On the other hand, the transmembrane domain significantly slowed down the ER to cis-Golgi transport and effectively counteracted the anterograde transport signal at this step. It may also take part in the retrieval of the protein, because the overall transport to the vacuole was more evidently slowed down. Consistently, the mutation of a conserved glutamine residue in the transmembrane domain further slowed down the transport in a step after arriving at the cis-Golgi. Taken together, the existence of the anterograde transport signal and the elements that regulate its function support the active recycling of Emp24p.
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PMID:Identification of potential regulatory elements for the transport of Emp24p. 984 83


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