The Arf-GTPase-activating protein Gcs1p is essential for sporulation and regulates the phospholipase D Spo14p

SPO14, encoding the major Saccharomyces cerevisiae phospholipase D (PLD), is essential for sporulation and mediates synthesis of the new membrane that encompasses the haploid nuclei that arise through meiotic divisions. PLD catalyzes the hydrolysis of phosphatidylcholine to phosphatidic acid (PA) and choline. PA stimulates Arf-GTPase-activating proteins (Arf-GAPs), which are involved in membrane trafficking events and actin cytoskeletal function. To determine if Spo14p-generated PA mediates its biological response through Arf-GAPs, we analyzed the sporulation efficiencies of cells deleted for each of the five known and potential yeast Arf-GAPs. Only gcs1delta mutants display a sporulation defect similar to that of spo14 mutants: cells deleted for GCS1 initiate the sporulation program but are defective in synthesis of the prospore membrane. Endosome-to-vacuole transport is also impaired in gcs1delta cells during sporulation. Furthermore, Arf-GAP catalytic activity, but not the pleckstrin homology domain, is required for both prospore membrane formation and endosome-to-vacuole trafficking. An examination of Gcs1p-green fluorescent protein revealed that it is a soluble protein. Interestingly, cells deleted for GCS1 have reduced levels of Spo14p-generated PA. Taken together, these results indicate that GCS1 is essential for sporulation and suggest that GCS1 positively regulates SPO14.     

GCS1, an Arf guanosine triphosphatase-activating protein in Saccharomyces cerevisiae, is required for normal actin cytoskeletal organization in vivo and stimulates actin polymerization in vitro

Recent cloning of a rat brain phosphatidylinositol 3,4, 5-trisphosphate binding protein, centaurin alpha, identified a novel gene family based on homology to an amino-terminal zinc-binding domain. In Saccharomyces cerevisiae, the protein with the highest homology to centaurin alpha is Gcs1p, the product of the GCS1 gene. GCS1 was originally identified as a gene conditionally required for the reentry of cells into the cell cycle after stationary phase growth. Gcs1p was previously characterized as a guanosine triphosphatase-activating protein for the small guanosine triphosphatase Arf1, and gcs1 mutants displayed vesicle-trafficking defects. Here, we have shown that similar to centaurin alpha, recombinant Gcs1p bound phosphoinositide-based affinity resins with high affinity and specificity. A novel GCS1 disruption strain (gcs1Delta) exhibited morphological defects, as well as mislocalization of cortical actin patches. gcs1Delta was hypersensitive to the actin monomer-sequestering drug, latrunculin-B. Synthetic lethality was observed between null alleles of GCS1 and SLA2, the gene encoding a protein involved in stabilization of the actin cytoskeleton. In addition, synthetic growth defects were observed between null alleles of GCS1 and SAC6, the gene encoding the yeast fimbrin homologue. Recombinant Gcs1p bound to actin filaments, stimulated actin polymerization, and inhibited actin depolymerization in vitro. These data provide in vivo and in vitro evidence that Gcs1p interacts directly with the actin cytoskeleton in S. cerevisiae.     

Dissecting the role of glutathione biosynthesis in Plasmodium falciparum

Glutathione (γ-glutamylcysteinyl-glycine, GSH) has vital functions as thiol redox buffer and cofactor of antioxidant and detoxification enzymes. Plasmodium falciparum possesses a functional GSH biosynthesis pathway and contains mM concentrations of the tripeptide. It was impossible to delete in P. falciparum the genes encoding γ-glutamylcysteine synthetase (γGCS) or glutathione synthetase (GS), the two enzymes synthesizing GSH, although both gene loci were not refractory to recombination. Our data show that the parasites cannot compensate for the loss of GSH biosynthesis via GSH uptake. This suggests an important if not essential function of GSH biosynthesis pathway for the parasites. Treatment with the irreversible inhibitor of γGCS L-buthionine sulfoximine (BSO) reduced intracellular GSH levels in P. falciparum and was lethal for their intra-erythrocytic development, corroborating the suggestion that GSH biosynthesis is important for parasite survival. Episomal expression of γgcs in P. falciparum increased tolerance to BSO attributable to increased levels of γGCS. Concomitantly expression of glutathione reductase was reduced leading to an increased GSH efflux. Together these data indicate that GSH levels are tightly regulated by a functional GSH biosynthesis and the reduction of GSSG.     

Some properties of glutathione biosynthesis-deficient mutants of Escherichia coli B

Mutants of Escherichia coli B that contain essentially no detectable glutathione were isolated. These mutants had a very low activity of gamma-glutamylcysteine synthetase or glutathione synthetase. No significant differences in growth in minimal medium were observed between the mutants and the parental strain. The mutants lacking gamma-glutamylcysteine synthetase activity were more susceptible to toxic compounds than either the parental strain or a glutathione synthetase-deficient strain. The mutants lacking gamma-glutamylcysteine synthetase activity were also susceptible to oxygen.     

Glutathione is required for growth and prespore cell differentiation in Dictyostelium

Glutathione (GSH) is the most abundant non-protein thiol in eukaryotic cells and acts as reducing equivalent in many cellular processes. We investigated the role of glutathione in Dictyostelium development by disruption of gamma-glutamylcysteine synthetase (GCS), an essential enzyme in glutathione biosynthesis. GCS-null strain showed glutathione auxotrophy and could not grow in medium containing other thiol compounds. The developmental progress of GCS-null strain was determined by GSH concentration contained in preincubated media before development. GCS-null strain preincubated with 0.2 mM GSH was arrested at mound stage or formed bent stalk-like structure during development. GCS-null strain preincubated with more than 0.5 mM GSH formed fruiting body with spores, but spore viability was significantly reduced. In GCS-null strain precultured with 0.2 mM GSH, prestalk-specific gene expression was delayed, while prespore-specific gene and spore-specific gene expressions were not detected. In addition, GCS-null strain precultured with 0.2 mM GSH showed prestalk tendency and extended G1 phase of cell cycle. Since G1 phase cells at starvation differentiate into prestalk cells, developmental defect of GCS-null strain precultured with 0.2 mM GSH may result from altered cell cycle. These results suggest that glutathione itself is essential for growth and differentiation to prespore in Dictyostelium.     

Glucosylceramides are critical for cell‐type differentiation and organogenesis,but not for cell viability in Arabidopsis

Glucosylceramides (GlcCer), glucose‐conjugated sphingolipids, are major components of the endomembrane system and plasma membrane in most eukaryotic cells. Yet the quantitative significance and cellular functions of GlcCer are not well characterized in plants and other multi‐organ eukaryotes. To address this, we examined Arabidopsis lines that were lacking or deficient in GlcCer by insertional disruption or by RNA interference (RNAi) suppression of the single gene for GlcCer synthase (GCS, At2g19880), the enzyme that catalyzes GlcCer synthesis. Null mutants for GCS (designated ‘gcs‐1’) were viable as seedlings, albeit strongly reduced in size, and failed to develop beyond the seedling stage. Heterozygous plants harboring the insertion allele exhibited reduced transmission through the male gametophyte. Undifferentiated calli generated from gcs‐1 seedlings and lacking GlcCer proliferated in a manner similar to calli from wild‐type plants. However, gcs‐1 calli, in contrast to wild‐type calli, were unable to develop organs on differentiation media. Consistent with a role for GlcCer in organ‐specific cell differentiation, calli from gcs‐1 mutants formed roots and leaves on media supplemented with the glucosylated sphingosine glucopsychosine, which was readily converted to GlcCer independent of GCS. Underlying these phenotypes, gcs‐1 cells had altered Golgi morphology and fewer cisternae per Golgi apparatus relative to wild‐type cells, indicative of protein trafficking defects. Despite seedling lethality in the null mutant, GCS RNAi suppression lines with ≤2% of wild‐type GlcCer levels were viable and fertile. Collectively, these results indicate that GlcCer are essential for cell‐type differentiation and organogenesis, and plant cells produce amounts of GlcCer in excess of that required for normal development.     

Up-regulation of the human gamma-glutamylcysteine synthetase regulatory subunit gene involves binding of Nrf-2 to an electrophile responsive element.

The rate-limiting step in the de novo synthesis of the cellular protectant glutathione is catalyzed by gamma-glutamylcysteine synthetase (GCS; also known as glutamine-L-cysteine ligase, GLCL), a heterodimer consisting of catalytic (GCS(h)) and regulatory (GCS(l)) subunits. Regulation of expression of the human gamma-glutamylcysteine synthetase regulatory subunit gene in response to beta-NF is mediated by an Electrophile Responsive Element (EpRE) [Moinova, H., and Mulcahy, R. T. (1998) J. Biol. Chem. 273, 14683-14689]. Oligonucleotide probes corresponding to wild-type and mutant EpRE sequences were used in gel-shift and super-shift analyses to identify proteins binding. Four protein:DNA complexes (a-d) with distinct mobilities were detected when the wild-type EpRE probe was incubated with nuclear extracts from control or beta-NF-treated HepG2 cells. Following beta-NF treatment, there was an increase in the intensity of a single band, band b. This band was eliminated in gel shifts employing mutant EpRE probes which abolish beta-NF inducibility, demonstrating a correlation between band b and transactivation. Super-shift analysis identified JunD, Nrf1, and Nrf2 in the EpRE-binding complexes. Antibodies to Nrf2 completely super-shifted the band b protein:DNA complex. These studies demonstrate that Nrf2 proteins recognize and bind the GCS(l) EpRE sequence to affect transactivation of the gene.     

Prenylated isoforms of yeast casein kinase I, including the novel Yck3p, suppress the gcs1 blockage of cell proliferation from stationary phase.

The GCS1 gene of the budding yeast Saccharomyces cerevisiae mediate the resumption of cell proliferation from the starved, stationary-phase state. Here we identify yeast genes that, in increased dosages, overcome the growth defect of gcs1 delta mutant cells. Among these are YCK1 (CK12) and YCK2 (CKI1), encoding membrane-associated casein kinase I, and YCK3, encoding a novel casein kinase I isoform. Some Yck3p gene product was found associated with the plasma membrane, like Yck1p and Yck2p, but most confractionated with the nucleus, like another yeast casein kinase I isoform, Hrr25p. Genetic studies showed that YCK3 and HRR25 constitute an essential gene family and that Yck3p can weakly substitute for Yck1p-Yck2p. For gcs1 delta suppression, both a protein kinase domain and a C-terminal prenylation motif were shown to be necessary. An impairment in endocytosis was found for gcs1 delta mutant cells, which was alleviated by an increased YCK2 gene dosage. The ability of an increased casein kinase I gene dosage to suppress the effects caused by the absence of Gcs1p suggests that Gcs1p and Yck1p-Yck2p affect parallel pathways.     

Role of glutathione in apoptosis induced by radiation as determined by 1H MR spectra of cultured tumor cells

The relationship between apoptosis induced by gamma radiation and glutathione in cells of two human cancer cell lines, HeLa from cervix carcinoma and MCF-7 from mammary carcinoma, was examined. MCF-7 cells appeared to be more radioresistant than HeLa cells, and radiation-induced apoptosis, which was monitored by assessing phosphatidylserine externalization, was observed in HeLa cells but not in MCF-7 cells. Glutathione levels monitored by (1)H MRS were higher in MCF-7 cells than in HeLa cells, while the opposite was true for the free glu signals. MCF-7 cells became more radiosensitive when treated with 0.1 mM buthionine sulfoximine, which inhibits GSH synthesis through inactivation of gamma-glutamylcysteine synthetase, with the concomitant appearance of radiation-induced apoptosis. We can thus reasonably associate, at least in part, the resistance of MCF-7 cells to apoptosis with a high level of glutathione and probably with a high activity of gamma-glutamylcysteine synthetase. A late decrease in glutathione concentration after irradiation was observed in MCF-7 cells, but not in HeLa cells and to a lesser degree in buthionine sulfoximine-treated MCF-7 cells. This would indicate that the radiation-induced decrease in glutathione concentration is not related to the onset of apoptosis, but it is more likely related to glutathione consumption as a result of detoxification reactions.     

Cloning and nucleotide sequence of a full-length cDNA for human liver gamma-glutamylcysteine synthetase.

We have cloned and sequenced a full-length cDNA for human liver gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in glutathione biosynthesis. The cDNA consists of 2634 bp containing an open reading frame encoding a protein of 367 amino acids and having a calculated M(r) = 72,773. The nucleotide sequence of the cDNA for human liver GCS shares an 84% overall similarity with the composite rat GCS sequence deduced from three overlapping partial cDNAs (Yan and Meister, JBC 265: 1588-1593, 1990). The deduced amino acid sequences are 94% similar. Comparison of Northern blots of total RNA isolated from rat kidney or liver with that from human kidney revealed the GCS mRNA to be larger in the human tissue (approximately 4.0 kb vs. approximately 3.7 kb). (The sequence for the human liver GCS cDNA has been assigned accession number M90656 in GenBank/EMBL databases.     

The Gcs1 Arf-GAP mediates Snc1,2 v-SNARE retrieval to the Golgi in yeast

Gcs1 is an Arf GTPase-activating protein (Arf-GAP) that mediates Golgi-ER and post-Golgi vesicle transport in yeast. Here we show that the Snc1,2 v-SNAREs, which mediate endocytosis and exocytosis, interact physically and genetically with Gcs1. Moreover, Gcs1 and the Snc v-SNAREs colocalize to subcellular structures that correspond to the trans-Golgi and endosomal compartments. Studies performed in vitro demonstrate that the Snc-Gcs1 interaction results in the efficient binding of recombinant Arf1Delta17N-Q71L to the v-SNARE and the recruitment of purified coatomer. In contrast, the presence of Snc had no effect on Gcs1 Arf-GAP activity in vitro, suggesting that v-SNARE binding does not attenuate Arf1 function. Disruption of both the SNC and GCS1 genes results in synthetic lethality, whereas overexpression of either SNC gene inhibits the growth of a distinct subset of COPI mutants. We show that GFP-Snc1 recycling to the trans-Golgi is impaired in gcs1Delta cells and these COPI mutants. Together, these results suggest that Gcs1 facilitates the incorporation of the Snc v-SNAREs into COPI recycling vesicles and subsequent endosome-Golgi sorting in yeast.     

Isolation of mutants of Schizosaccharomyces pombe unable to synthesize cadystin, small cadmium-binding peptides

Schizosaccharomyces pombe synthesize small cadmium-binding peptides cadystin, structure of which is (gamma-Glu-Cys)n-Gly, in response to cadmium. Mutants unable to synthesize cadystin were found in the mutants hypersensitive to cadmium. Some of them lack activity of either gamma-glutamylcysteine synthetase (EC 6.3.2.2) or glutathione synthetase (EC 6.3.2.3), enzyme involved in glutathione biosynthesis. Some mutants have the same activity levels of these enzymes as wild type has. These results indicate that some steps of cadystin biosynthesis are catalyzed by the enzymes catalyzing glutathione biosynthesis.     

Regulation of the gene encoding glutathione synthetase from the fission yeast

The fission yeast cells that contained the cloned glutathione synthetase (GS) gene showed 1.4-fold higher glutathione (GSH) content and 1.9-fold higher GS activity than the cells without the cloned GS gene. Interestingly, gamma-glutamylcysteine synthetase activity increased 2.1-fold in the S. pombe cells that contained the cloned GS gene. The S. pombe cells that harbored the multicopy-number plasmid pRGS49 (containing the cloned GS gene) showed a higher level of survival on solid media with cadmium chloride (1 mM) or mercuric chloride (10 microM) than the cells that harbored the YEp357R vector. The 506 bp upstream sequence from the translational initiation point and N-terminal 8 amino acid-coding region were fused into the promoterless beta-galactosidase gene of the shuttle vector YEp367R to generate the fusion plasmid pUGS39. Synthesis of beta-galactosidase from the fusion plasmid pUGS39 was significantly enhanced by cadmium chloride and NO-generating S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside (SN). It was also induced by L-buthionine-(S,R)-sulfoximine, a specific inhibitor of gamma-glutamylcysteine synthetase (GCS). We also found that the expression of the S. pombe GS gene is regulated by the Atf1-Spc1-Wis1 signal pathway.     

Knockdown of glutamate-cysteine ligase by small hairpin RNA reveals that both catalytic and modulatory subunits are essential for the survival of primary neurons

Glutathione deficiency is an early biochemical feature that occurs during apoptotic neuronal death associated with certain neurological disorders such as Parkinson disease. However, whether specific targeting of glutathione biosynthesis in neurons is sufficient to trigger neurodegeneration remains undetermined. To address this issue, we used a vector-based small hairpin RNA (shRNA) strategy to knock down each subunit of glutamate-cysteine ligase (GCL; gamma-glutamylcysteine synthetase), the heterodimeric enzyme that catalyzes the rate-limiting step of glutathione biosynthesis. Independent targeting of the catalytic and modulatory subunits by shRNA caused disruption of GCL as assessed by Northern and Western blotting, enzyme activity, and glutathione concentrations. Silencing each subunit in primary cortical neurons spontaneously elicited time-dependent apoptotic death, an effect that was synergistic with glutamate or nitric oxide treatment. Moreover, neuronal apoptosis by GCL knockdown was rescued by expressing the corresponding subunit full-length cDNA carrying silent mutations within the shRNA target cDNA sequence and by incubating neurons with gamma-glutamylcysteine or glutathione ethyl ester. In contrast, supplying glutathione precursors to neurons from co-cultured astrocytes did not prevent the apoptotic death triggered by GCL knockdown. Finally, overexpressing the catalytic (but not modulatory) GCL subunit full-length cDNA increased enzyme activity and glutathione concentrations, yielding neurons more resistant to glutamate- or nitric oxide-mediated apoptosis. Thus, specific and independent disruption of each subunit of GCL in neurons can be said to cause a primary decrease in glutathione that is sufficient to promote neurodegeneration.     

Cysteine regulates expression of cysteine dioxygenase and gamma-glutamylcysteine synthetase in cultured rat hepatocytes

Rat hepatocytes cultured for 3 days in basal medium expressed low levels of cysteine dioxygenase (CDO) and high levels of gamma-glutamylcysteine synthetase (GCS). When the medium was supplemented with 2 mmol/l methionine or cysteine, CDO activity and CDO protein increased by >10-fold and CDO mRNA increased by 1.5- or 3.2-fold. In contrast, GCS activity decreased to 51 or 29% of basal, GCS heavy subunit (GCS-HS) protein decreased to 89 or 58% of basal, and GCS mRNA decreased to 79 or 37% of basal for methionine or cysteine supplementation, respectively. Supplementation with cysteine consistently yielded responses of greater magnitude than did supplementation with an equimolar amount of methionine. Addition of propargylglycine to inhibit cystathionine gamma-lyase activity and, hence, cysteine formation from methionine prevented the effects of methionine, but not those of cysteine, on CDO and GCS expression. Addition of buthionine sulfoximine to inhibit GCS, and thus block glutathione synthesis from cysteine, did not alter the ability of methionine or cysteine to increase CDO. GSH concentration was not correlated with changes in either CDO or GCS-HS expression. The effectiveness of cysteine was equivalent to or greater than that of its precursors (S-adenosylmethionine, cystathionine, homocysteine) or metabolites (taurine, sulfate). Taken together, these results suggest that cysteine itself is an important cellular signal for upregulation of CDO and downregulation of GCS.     

Flavonoids increase the intracellular glutathione level by transactivation of the gamma-glutamylcysteine synthetase catalytical subunit promoter

Fruits and vegetables protect against cancer by so far not well-characterized mechanisms. One likely explanation for this effect is that dietary plants contain substances able to control basic cellular processes such as the endogenous defense against oxidative stress. Oxidative stress is pivotal in many pathological processes and reduced oxidative stress is implicated in prevention of disease. Our results demonstrate that extract from onion and various flavonoids induce the cellular antioxidant system. Onion extract and quercetin were able to increase the intracellular concentration of glutathione by approximately 50%. Using a reporter construct where reporter expression is driven by the gamma-glutamylcysteine synthetase (GCS) heavy subunit (GCS(h)) promoter we show that onion extract, quercetin, kaempferol, and apigenin increased reporter gene activity, while a fourth flavonoid, myricetin and sugar conjugates of quercetin were unable to increase reporter expression. Quercetin was also able to induce a distal part of the GCS(h) promoter containing only two antioxidant-response/electrophile-response elements (ARE/EpRE). Our data strongly suggest that flavonoids are important in the regulation of the intracellular glutathione levels. This effect may be exerted in part through GCS gene regulation, and may also contribute to the disease-preventing effect of fruits and vegetables.