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)

Sec4, a GTP-binding protein of the ras superfamily, is required for exocytosis in the budding yeast Saccharomyces cerevisiae. To test the role of GTP hydrolysis in Sec4 function, we constructed a mutation, Q-79----L, analogous to the oncogenic mutation of Q-61----L in Ras, in a region of Sec4 predicted to interact with the phosphoryl group of GTP. The sec4-leu79 mutation lowers the intrinsic hydrolysis rate to unmeasurable levels. A component of a yeast lysate specifically stimulates the hydrolysis of GTP by Sec4, while the rate of hydrolysis of GTP by Sec4-Leu79 can be stimulated by this GAP activity to only 30% of the stimulated hydrolysis rate of the wild-type protein. The decreased rate of hydrolysis results in the accumulation of the Sec4-Leu79 protein in its GTP-bound form in an overproducing yeast strain. The sec4-leu79 allele can function as the sole copy of sec4 in yeast cells. However, it causes recessive, cold-sensitive growth, a slowing of invertase secretion, and accumulation of secretory vesicles and displays synthetic lethality with a subset of other secretory mutants, indicative of a partial loss of Sec4 function. While the level of Ras function reflects the absolute level of GTP-bound protein, our results suggest that the ability of Sec4 to cycle between its GTP and GDP bound forms is important for its function in vesicular transport, supporting a mechanism for Sec4 function which is distinct from that of the Ras protein.
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PMID:Hydrolysis of GTP by Sec4 protein plays an important role in vesicular transport and is stimulated by a GTPase-activating protein in Saccharomyces cerevisiae. 156 38

Core glycosylated proteins formed in the yeast endoplasmic reticulum (ER) are transported to the Golgi body, where oligosaccharides are elongated by addition of outer-chain carbohydrate. The transport process is blocked in a temperature-sensitive secretion mutant (sec18) of Saccharomyces cerevisiae, which accumulates core glycosylated invertase (product of SUC2; EC 3.2.1.26) in the ER. To approach the molecular mechanism of this transport process, we have devised a reaction in which core glycosylated invertase, accumulated in sec18 cells, is transferred to the Golgi body in vitro. For this purpose, membranes from sec18, SUC2 cells that are also defective in an outer chain alpha-1----3-mannosyltransferase (mnnl) are mixed with membranes from a strain that contains the transferase but is deficient in invertase (MNNl, delta SUC2). Transfer is detected by the acquisition of outer-chain alpha-1----3-linked mannose residues dependent on both donor and recipient membranes. The reaction is temperature and detergent sensitive and requires ATP, GDP-mannose, Mg2+, and Mn2+, and the product invertase remains associated with sedimentable membranes. Treatment of donor, but not acceptor, membranes with N-ethylmaleimide or trypsin inactivates transfer competence. These characteristics suggest that the ER, or a vesicle derived from the ER, contributes invertase to a chemically distinct compartment where mannosyl modification is executed.
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PMID:Interorganelle transfer and glycosylation of yeast invertase in vitro. 242 Dec 86

When incubated at a restrictive temperature, Saccharomyces cerevisiae sec59 mutant cells accumulate inactive and incompletely glycosylated forms of secretory proteins. Three different secretory polypeptides (invertase, pro-alpha-factor, and pro-carboxypeptidase Y) accumulated within a membrane-bounded organelle, presumably the endoplasmic reticulum, and resisted proteolytic degradation unless the membrane was permeabilized with detergent. Molecular cloning and DNA sequence analysis of the SEC59 gene predicted an extremely hydrophobic protein product of 59 kilodaltons. This prediction was confirmed by reconstitution of the sec59 defect in vitro. The alpha-factor precursor, which was translated in a soluble fraction from wild-type cells, was translocated into, but inefficiently glycosylated within, membranes from sec59 mutant cells. Residual glycosylation activity of membranes of sec59 cells was thermolabile compared with the activity of wild-type membranes. Partial restoration of glycosylation was obtained in reactions that were supplemented with mannose or GDP-mannose, but not those supplemented with other sugar nucleotides. These results were consistent with a role for the Sec59 protein in the transfer of mannose to dolichol-linked oligosaccharide.
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PMID:Sec59 encodes a membrane protein required for core glycosylation in Saccharomyces cerevisiae. 265 87

The yeast Saccharomyces cerevisiae X2180 strain with the mnn1 mnn2 mnn9 mutations, all of which affect mannoprotein glycosylation, synthesizes N-linked oligosaccharides having the following structure: (Formula: see text) whereas the mnn1 mnn2 mutant extends the alpha 1----6-linked backbone of some of the core oligosaccharides by adding 20-30 mannose units. Membrane fractions from the mnn1 mnn2 and mnn1 mnn2 mnn9 mutants are equally effective in catalyzing transfer from GDP-[3H]mannose to add mannose in both alpha 1----2 and alpha 1----6 linkages to an oligosaccharide having the following structure: (Formula: see text) but neither membrane preparation can utilize the homologous mnn1 mnn2 mnn9 oligosaccharide as an acceptor. Thus, addition of the alpha 1----2-linked mannose side chain to the terminal alpha 1----6-linked mannose in oligosaccharides of the mnn9 mutant inhibits the elongation reaction and may serve as an important structural control of mannoprotein glycosylation. The mnn9 mutation also increases the transit time for invertase secretion, meaning that this mutation could affect the processing machinery in the Golgi apparatus.
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PMID:Regulation of the protein glycosylation pathway in yeast: structural control of N-linked oligosaccharide elongation. 332 Oct 55

Epithelial cells of the rat small intestine were collected as a gradient of villus to crypt cells. Homogenates of these cells incubated with GDP-D-[14C]mannose in the presence of MnCl2 incorporated radioactivity into dolichyl mannosyl phosphate and a mixutre of dolichyl pyrophosphate oligosaccharides varying in the size of their oligosaccharide moiety. The labeled oligosaccharides formed in villus cell homogenates appeared shorter than those formed in crypt cell homogenates. The addition of dolichyl phosphate greatly stimulated the synthesis of dolichyl mannosyl phosphate. The initial rate of synthesis of dolichyl mannosyl phosphate from GDP-D-[14C]mannose and exogenous dolichyl phosphate was highest in an intermediate cell fraction having a low specific activity of sucrase and alkaline phosphatase and an intermediate specific activity of thymidine kinase. To compare the rates of dolichyl mannosyl phosphate synthesis in the different cell fractions, it was essential to control degradation of GDP-D-[14]mannose by the addition of AMP to the incubation, since villus cells degraded GDP-D-[14C]mannose much faster than crypt cells.
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PMID:Glycoprotein biosynthesis in intestinal epithelial cells during differentiation. Incorporation of [14C]mannose from GDP-[14C]mannose into dolichol derivatives. 615 73

We have previously demonstrated the appearance of fucosyl asialo-GM1 (FGA1) in the small-intestinal epithelial cells of germ-free mice via the induction of GDP-fucose: asialo-GM1 (GA1) alpha(1 leads to 2) fucosyltransferase (FT) after the conventionalization of these animals (Umesaki Y, Sakata T, Yajima T: Biochem Biophys Res Commun 105:439, 1982). The present study, based on this earlier work, demonstrates the changes in the glycolipid antigens of the small-intestinal epithelial-cell membrane as shown immunohistochemically with specific antibodies raised against asialo GM1 (GA1) and FGA1. In germ-free mice, GA1 was localized both in the villus cells and in the crypt cells. In the process of conventionalization, FGA1 appeared in the villus cells while the GA1 content of these cells was decreased. Four to 5 days after the conventionalization procedure, the fluorescence produced by anti-FGA1 was strongest in the villus cells, while that produced by anti-GA1 was detected only in the crypt cells. At this same time the FT activity of the small-intestinal mucosa was highest, with most of the GA1 apparently being converted into FGA1, as shown in the paper cited above. Thereafter, the GA1 content of both the villus and crypt cells again increased greatly. On the other hand, the fluorescence produced with anti-FGA1 decreased, and could no longer be detected 14 days after conventionalization. The activity of FT, measured biochemically in epithelial cells differentially isolated from the villus tip to the crypt, was greater in the villus than in the crypt region. This confirmed the intense staining with anti-FGA1 that was seen in villus cells. The fluorescence produced by the two anti-glycolipid antibodies used in the study distributed not only in the microvillus membrane but also to some extent in the basolateral membrane. The localization of the respective glycolipids contrasted with that of the glycoprotein sucrase--isomaltase enzyme complex, the fluorescence of which was exclusively confined to the microvillus-membrane side of the villus cells.
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PMID:Immunohistochemical and biochemical demonstration of the change in glycolipid composition of the intestinal epithelial cell surface in mice in relation to epithelial cell differentiation and bacterial association. 669 58

A [3H]mannose suicide selection has been used to isolate mutants in yeast which contain temperature-sensitive defects in asparagine-linked glycosylation. The surviving cells were screened at the nonpermissive temperature for a decreased ability to incorporate [3H]mannose and for defects in glycosylation of the secreted protein invertase. One of these mutants (alg1-1) has been characterized and found to be blocked in the assembly of the lipid-linked oligosaccharide precursor. The alg1-1 cells synthesize mannosyl compounds at 60% of the wild type level at the nonpermissive temperature and 105% of the wild type level at the permissive temperature. In vivo labeling experiments have demonstrated that alg1-1 cells are able to synthesize GlcNAc2-lipid but are unable to synthesize any mannose-containing oligosaccharide-lipids. This result was confirmed by in vitro labeling of yeast membranes. When incubated with UDP-[3H]GlcNAc, alg1-1 membranes synthesized GlcNAc2-lipid but failed to elongate it when GDP-Man was added. The alg1-1 membranes also failed to elongate exogenous GlcNAc2-lipid but were able to convert Man1GlcNAc2-lipid to Man5-Glc-NAc2-lipid in the presence of GDP-Man. These results indicate that the alg1-1 mutant is blocked specifically in the addition of the first mannose residue to the lipid-linked oligosaccharide precursor.
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PMID:Temperature-sensitive yeast mutants deficient in asparagine-linked glycosylation. 703 80

Current models for nucleotide sugar use in the Golgi apparatus predict a critical role for the lumenal nucleoside diphosphatase. After transfer of sugars to endogenous macromolecular acceptors, the enzyme converts nucleoside diphosphates to nucleoside monophosphates which in turn exit the Golgi lumen in a coupled antiporter reaction, allowing entry of additional nucleotide sugar from the cytosol. To test this model, we cloned the gene for the S. cerevisiae guanosine diphosphatase and constructed a null mutation. This mutation should reduce the concentrations of GDP-mannose and GMP and increase the concentration of GDP in the Golgi lumen. The alterations should in turn decrease mannosylation of proteins and lipids in this compartment. In fact, we found a partial block in O- and N-glycosylation of proteins such as chitinase and carboxypeptidase Y and underglycosylation of invertase. In addition, mannosylinositolphosphorylceramide levels were drastically reduced.
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PMID:Guanosine diphosphatase is required for protein and sphingolipid glycosylation in the Golgi lumen of Saccharomyces cerevisiae. 839 37

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

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


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