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 previously isolated glycopeptides derived from yeast invertase that acted as highly potent elicitors in suspension-cultured tomato cells, inducing ethylene biosynthesis and phenylalanine ammonia-lyase activity, and we have found that the high mannose oligosaccharides released from the pure glycopeptide elicitors by endo-beta-N-acetylglucosaminidase H acted as suppressors of elicitor activity (Basse, C. W., Bock, K., and Boller, T. (1992) J. Biol. Chem. 267, 10258-10265). One of the elicitor-active glycopeptides (gp 8c) was labeled with t-butoxycarbonyl-L-[35S]methionine and purified by reversed phase high performance liquid chromatography resulting in a specific radioactivity of the derivative of about 900 Ci/mmol. This radiolabeled glycopeptide showed specific, saturable, and reversible binding to whole tomato cells under conditions in which cells are responsive to elicitors as well as to microsomal membranes derived from these cells. Ligand saturation experiments, performed with microsomal membranes, gave a dissociation constant (Kd) of 3.3 nM as determined by Scatchard analysis. Various glycopeptide elicitors and preparations from yeast invertase were compared with respect to their abilities to compete for binding of 35S-labeled gp 8c to tomato membranes and to induce ethylene biosynthesis in tomato cells. These studies revealed a high degree of correlation between elicitor activities in vivo and displacement activities in vitro. In both tests, a high activity depended on the presence of glycan side chains consisting of more than 8 mannosyl residues. The high mannose oligosaccharides that acted as suppressors of elicitor activity in vivo competed for binding of the labeled elicitor also. The suppressor-active glycan Man11GlcNAc and the elicitor-active gp 8c exhibited very similar displacement activities, and the inhibitory constant (Ki) of the glycan Man11GlcNAc was very similar to the Kd value calculated for 35S-labeled gp 8c, indicating that the glycopeptide elicitors and the glycan suppressors derived from these elicitors competed with similar affinities for the same binding site. The suppressor-inactive glycan Man8GlcNAc had a 200-fold lower capacity to compete for binding of 35S-labeled gp 8c to tomato membranes compared with the suppressor-active glycan Man11GlcNAc. Our results demonstrate the existence of a specific elicitor binding site in tomato cell membranes and suggest that glycopeptides and glycans act as agonists and antagonists for induction of the stress response, respectively, by competing for this binding site.
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PMID:High affinity binding of a glycopeptide elicitor to tomato cells and microsomal membranes and displacement by specific glycan suppressors. 832 50

In the yeast Golgi compartments, at least five, and potentially several additional mannosyltransferases are involved in elongating to 'mannan' the core Man8GlcNAc2 oligosaccharide trimmed from Glc3Man9GlcNAc2 in the endoplasmic reticulum. Structural studies on oligosaccharides from alg3 mutant yeast, which lack the four upper arm mannoses donated by Man-P-Dol (where Dol is dolichol), verified that the new alpha 1,6-branch in endo H-resistant mannan in this strain is efficiently initiated in vivo on the alpha 1,3-linked core residue of the lipid-oligosaccharide form of Man5GlcNAc2 (Verostek et al., J. Biol. Chem., 266, 5547-5551, 1991). This Man5GlcNAcGlcNAc[3H]ol isomer (where GlcNAc[3H]ol is N-acetylglucosamin [1-3H] itol) was found to be an excellent acceptor for a number of GDP-Man-dependent Golgi mannosyltransferases in detergent-solubilized yeast membrane preparations: an alpha 1,3-mannosyltransferase (Mnn1p), an alpha 1,6-mannosyltransferase (Och1p) and two alpha 1,2-mannosyltransferases (Mnt1p/Kre2p,?) whose products were readily identified by 1H NMR spectroscopy. The Man6GlcNAcGlcNAc[3H]ol isomers formed were easily defined by alpha 1,2-mannosidase sensitivity and either Bio-Gel P-4 gel filtration or AX-5 high-performance liquid chromatography. In general, mannosyltransferases present in detergent-solubilized microsomes from most yeast strains mimicked the array of sugar linkages observed on their respective glycoproteins. However, in the case of the Saccharomyces pmr1 mutant, an alpha 1,3-mannosyltransferase was active in microsomal extracts, but the alpha 1,3-Man epitope could not be identified on Western blots of cellular glycoproteins using sugar linkage-specific antibodies or lectins. The in vitro transferase assay is simple, rapid and accurate, and in the case of pmr1 suggests that in vivo either invertase is misrouted during secretion or the alpha 1,3-mannosyltransferase is mistargeted after its synthesis in this mutant.
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PMID:Mannosyltransferase activities in membranes from various yeast strains. 860 69

A full-length cDNA for NADPH-cytochrome P450 reductase from Candida maltosa was cloned and sequenced. The derived amino acid sequence showed a high similarity to the reductases from other eukaryotes. Expression in Saccharomyces cerevisiae under control of the GAL10 promoter resulted in an approximately 70-fold increase in NADPH-cytochrome c reductase activity in the microsomal fraction. The functional integrity of the heterologously expressed reductase as an electron transfer component for alkane hydroxylating cytochrome P450 from C. maltosa was shown in a reconstituted system containing both enzymes in a highly purified state. The signal-anchor sequence of the reductase was identified within the N-terminal region of the protein by means of constructing and expressing fusion proteins with the cytosolic form of yeast invertase. The first 33 amino acids turned out to be sufficient for stable membrane insertion, wild-type membrane orientation and retention in the endoplasmic reticulum. As shown by immunoelectron microscopy, the heterologously expressed reductase was integrated into the endoplasmic reticulum of the host organism. It triggered a strong proliferation of the membrane system. This membrane-inducing property of the reductase was transferable to the cytosolic reporter protein with the same N-terminal sequences that confer membrane insertion.
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PMID:Candida maltosa NADPH-cytochrome P450 reductase: cloning of a full-length cDNA, heterologous expression in Saccharomyces cerevisiae and function of the N-terminal region for membrane anchoring and proliferation of the endoplasmic reticulum. 870 6

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

To further understand how membrane proteins are sorted in the secretory system, we devised a strategy that involves the expression of a membrane-anchored yeast invertase in transgenic plants. The construct consisted of a signal peptide followed by the coding region of yeast invertase and the transmembrane domain and cytoplasmic tail of calnexin. The substitution of a lysine near the C terminus of calnexin with a glutamic acid residue ensured progression through the secretory system rather than retention in or return to the endoplasmic reticulum. In the transformed plants, invertase activity and a 70-kD cross-reacting protein were found in the vacuoles. This yeast invertase had plant-specific complex glycans, indicating that transport to the vacuole was mediated by the Golgi apparatus. The microsomal fraction contained a membrane-anchored 90-kD cross-reacting polypeptide, but was devoid of invertase activity. Our results indicate that this membrane-anchored protein proceeds in the secretory system beyond the point where soluble proteins are sorted for secretion, and is detached from its membrane anchor either just before or just after delivery to the vacuole.
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PMID:Delivery of a secreted soluble protein to the vacuole via a membrane anchor. 1044 79

We study here the binding of ribosomes to the endoplasmic reticulum (ER) membrane and its dependence on nascent polypeptide-associated complex (NAC). For this, we use an in vitro translation system in combination with isolated microsomes. Importantly, all components in the system are derived from a single source, Saccharomyces cerevisiae. Ribosome nascent chains (RNCs) of the two naturally occurring invertase species (secreted or cytosolic) were prepared in wild-type, delta alpha NAC or delta alpha beta 1 beta 3 NAC translation lysates and tested for binding to the corresponding microsomal membranes. We provide evidence that NAC prevents binding of RNCs without a signal sequence to yeast membranes. In the absence of NAC, signal-less RNCs are able to bind to ER membranes. However, following puromycin treatment, only very few nascent chains translocate into the lumen, as detected by glycosylation.
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PMID:The nascent polypeptide-associated complex (NAC) of yeast functions in the targeting process of ribosomes to the ER membrane. 1051 32

The sodium-D-glucose cotransporter (SGLT1) was expressed in a yeast mutant strain NY 17 (sec6-4) that accumulates secretory vesicles at a nonpermissive temperature because of a block in the delivery of these vesicles to the plasma membrane. By differential centrifugation a microsomal fraction enriched in secretory vesicles was prepared with a high specific activity of the vanadate-sensitive H+-ATPase and invertase. In this membrane fraction one protein band of an apparent molecular weight of 55 kDa representing the nonglycosylated SGLT1 protein could be detected by immunochemical analysis. In addition, higher molecular weight protein bands probably representing dimers and aggregates were found. In transport studies with the microsomes D-glucose fluxes showed asymmetric properties: efflux experiments revealed the typical properties of the SGLT1 such as sodium dependence, inhibition by phlorizin and potential dependence. Influx of D-glucose showed no dependence on sodium and was not inhibited by phlorizin. Furthermore, the transporter exhibited a striking asymmetry with regard to the D-glucose affinity and the sugar specificity. These results suggest that the orientation of the SGLT1 expressed in yeast secretory vesicles is, indeed, inverted with regard to its configuration in the plasma membrane of epithelial cells. Moreover, there are striking functional differences between the periplasmic and cytoplasmic face of the transporter.
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PMID:Functional asymmetry of the sodium-D-glucose cotransporter expressed in yeast secretory vesicles. 1122 Mar 64

The Golgi plays a fundamental role in posttranslational glycosylation, transport, and sorting of proteins. The mechanism of protein transport through the Golgi has been seen as controversial in recent years. During the characterization of N-glycosylation-defective mutants (ngd) previously isolated by this laboratory, it was found that ngd20 is allelic to sec20. SEC20 was reported to be required for transport from endoplasmic reticulum to Golgi, but its precise function remains to be determined. We show now that SEC20 is also required for N- and O-glycosylation in the Golgi but not in the ER, in a cargo-specific manner, and that the glycosylation defect does not correlate with the secretory defect. By pulse-chase labeling experiments in combination with mannose linkage-specific antibodies, invertase and carboxypeptidase were found to be efficiently secreted to their final compartment, even upon shift to the nonpermissive temperature, while glycosylation in the Golgi was severely impaired. Using microsomal membranes isolated from ngd20, we found that mannosyl transfer from GDP-Man to various mannose-oligosaccharides, indicative for Golgi mannosylation, was strongly diminished. Analysis of the carbohydrate component of chitinase, an exclusively O-mannosylated protein, or of the bulk mannoprotein indicates that O-mannosylation is also reduced. The results demonstrate that in addition to secretion SEC20 also affects glycosylation in the Golgi, presumably because it exerts a more general role in maintenance and function of the Golgi compartments.
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PMID:The yeast SEC20 gene is required for N- and O-glycosylation in the Golgi. Evidence that impaired glycosylation does not correlate with the secretory defect. 1147 10

Purified glycopeptides derived from yeast invertase act as highly potent elicitors in suspension-cultured tomato (Lycopersicon esculentum [L.] Mill) cells, inducing ethylene biosynthesis and phenylalanine ammonia lyase half-maximally at concentrations of 1 to 5 nM. We previously demonstrated the presence of a high-affinity binding site that specifically recognized these glycopeptides in cells and microsomal membranes of tomato (C.W. Basse, A. Fath, T. Boller [1993] J Biol Chem 268: 14724-14731). This elicitor-binding site was solubilized in an active form from the microsomal membranes using the neutral detergents n-dodecylmaltoside and n-dodecanoylsucrose and purified 67-fold in a single step by anion-exchange chromatography. Ligand saturation studies and competition experiments with unlabeled glycopeptides and glycans demonstrated that the detergent-solubilized elicitor-binding site retained the high affinity (Kd approximately 1-4 nM) and selectivity of the membrane-bound form. The binding site was found to have a high affinity for N-linked glycans with nine mannosyl residues from fungal glycoproteins, whereas it did not recognize the typical mammalian glycans with nine mannosyl residues, demonstrating further its high selectivity.
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PMID:Solubilization, Partial Purification, and Characterization of a Binding Site for a Glycopeptide Elicitor from Microsomal Membranes of Tomato Cells. 1222 70

In an in vitro system comprising a yeast cell-free translation system, yeast microsomes and mRNA encoding prepro-alpha-factor, the translocation of this protein across the membrane of the microsomal vesicle and its glycosylation could b uncoupled from its translation. Such post-translational processing is dependent upon the presence of ATP in the system. It is not, however, affected by a variety of uncouplers, ionophores or inhibitors, including carbonyl cyanide m-chlorophenyl hydrazone (CCCP), valinomycin, nigericin, dinitrophenol (DNP), potassium cyanide (KCN) or N-ethyl maleimide (NEM). This mechanism of translocation is significant as it indicates that a protein of 18 000 daltons is capable of crossing an endoplasmic reticulum-derived membrane post-translationally. For the moment, this phenomenon seems to be restricted to prepro-alpha-factor in the yeast in vitro system. Neither invertase nor IgG chi light chain could be translocated post-translationally in yeast, nor was such processing observed for prepro-alpha-factor in a wheat germ system supplemented with canine pancreatic microsomes.
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PMID:Secretion in yeast: translocation and glycosylation of prepro-alpha-factor in vitro can occur via an ATP-dependent post-translational mechanism. 1595 17


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