Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M101451200 on August 10, 2001

J. Biol. Chem., Vol. 276, Issue 41, 37794-37801, October 12, 2001
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
276/41/37794    most recent
M101451200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Masuda, C. A.
Right arrow Articles by Montero-Lomelí, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Masuda, C. A.
Right arrow Articles by Montero-Lomelí, M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Phosphoglucomutase Is an in Vivo Lithium Target in Yeast*

Claudio A. MasudaDagger , Marcelle A. XavierDagger , Katherine A. Mattos§||, Antonio GalinaDagger , and Mónica Montero-LomelíDagger

From the Dagger  Departamento de Bioquímica Médica, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, C. P. 68041, Rio de Janeiro, RJ, 21941-590, Brazil and § Laboratório de Glicobiologia, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, 21949-900, Brazil

Received for publication, February 14, 2001, and in revised form, July 26, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Lithium is a drug frequently used in the treatment of manic depressive disorder. We have observed that the yeast Saccharomyces cerevisiae is very sensitive to lithium when growing in galactose medium. In this work we show that lithium inhibits with high affinity yeast (IC50 ~ 0.2 mM) and human (IC50 ~ 1.5 mM) phosphoglucomutase, the enzyme that catalyzes the reversible conversion of glucose 1-phosphate to glucose 6-phosphate. Lithium inhibits the rate of fermentation when yeast are grown in galactose and induces accumulation of glucose 1-phosphate and galactose 1-phosphate. Accumulation of these metabolites was also observed when a strain deleted of the two isoforms of phosphoglucomutase was incubated in galactose medium. In glucose-grown cells lithium reduces the steady state levels of UDP-glucose, resulting in a defect on trehalose and glycogen biosynthesis. Lithium acts as a competitive inhibitor of yeast phosphoglucomutase activity by competing with magnesium, a cofactor of the enzyme. High magnesium concentrations revert lithium inhibition of growth and phosphoglucomutase activity. Lithium stress causes an increase of the phosphoglucomutase activity due to an induction of transcription of the PGM2 gene, and its overexpression confers lithium tolerance in galactose medium. These results show that phosphoglucomutase is an important in vivo lithium target.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The monovalent cation lithium has been widely used in the treatment of manic depressive disorder (1, 2). Despite its frequent clinical use, the molecular mechanism underlying its action is poorly understood. Some of the hypotheses implicate central cellular signal transduction pathways (3, 4). One model is the "inositol depletion hypothesis" (5), which is based on the inhibition of the inositol monophosphatases (IMPases)1 (6, 7) and inositol polyphosphate 1-phosphatases (IPPases) (8) by lithium. This inhibition would lead to the impairment of the inositol 1,4,5-triphosphate-dependent signal transduction pathway due to depletion of cellular myo-inositol (5). Another enzyme involved in signal transduction inhibited by lithium is the glycogen synthase kinase-3beta (9, 10). This kinase is part of the wnt signaling pathway, which is involved in the developmental process of many different organisms (11). In fact, lithium inhibits the correct development of Xenopus laevis embryos (9, 12), sea urchin (13), Dictyostelium discoideum (14), and Tetrahymena thermophila (15). These theratogenic effects are proposed to be due to the inhibition of the glycogen synthase kinase-3beta (9, 10) since loss of function of this enzyme produces the same phenotypes in D. discoideum (16) and Xenopus (17-19). Additionally, other enzymes not related directly to signal transduction are known to be inhibited by lithium such as fructose 1,6-bisphosphatase (FBPase) from rabbit (20) and mouse (21), rabbit phosphoglucomutase (PGM) (22, 23), and the 3'(2')-5'-bisphosphate nucleotidases (BPNases) from yeast (24) and humans (25, 26). Interestingly, it was shown that IMPases, IPPases, FBPases, and BPNases share a structural motif responsible for lithium binding (27).


View larger version (21K):
[in this window]
[in a new window]
  		Fig. 1.   Schematic representation of the initial steps of the galactose and glucose metabolism pathways. GALT, galactose-1-phosphate uridyltransferase; GALE, UDP-glucose 4 epimerase; HXK, hexokinase; GLK, glucokinase. Other abbreviations are defined in Footnote 1. Arrows indicates the flux in the sugar catabolism direction. However, all reactions represented are reversible.

We have used Saccharomyces cerevisiae as an eukaryotic cell model to study mechanisms of lithium action. We have observed previously (28) that lithium is very toxic to yeast cells growing in medium with galactose as the sole carbon source. Galactose is metabolized in yeast, as in humans, by the Leloir pathway (29-31). This pathway (Fig. 1) consists of a series of reactions catalyzed by galactokinase (GK), galactose 1-phosphate uridyltransferase (GALT), and UDP-galactose 4-epimerase (GALE) that transform galactose to glucose 1-phosphate (Glc-1-P). In yeast, the GAL1, GAL7, and GAL10 genes encode these enzymes, respectively (32, 33). Then, PGM, which is encoded in yeast by the genes GAL5/PGM2 and PGM1 (34-36), converts the Glc-1-P to glucose 6-phosphate (Glc-6-P), which is further metabolized through glycolysis (Fig. 1). The genes GAL1, GAL7, GAL10, and GAL5, together with GAL2 gene, which encodes a galactose permease (37), are regulated by the transcription factor Gal4p and the transcription regulator Gal80p, being induced by galactose and repressed by glucose (35, 38, 39). Mutations that affects any of these enzymes causes a growth deficiency in galactose (32-34, 37).

In this work we have studied the effects of lithium on galactose metabolism in yeast. We present evidences that PGM is an in vivo target of lithium responsible for the galactose-specific lithium toxicity. We also show that human PGM activity is inhibited by lithium. Possible involvement of PGM in the mechanism of lithium action in humans is discussed.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Strains and Plasmid Construction-- Escherichia coli strain XL1-Blue was used for plasmid construction. S. cerevisiae JF291 (Matalpha , leu2, 3-112, his3-1, ura3-52, PGM1, PGM2) and JF645 (Matalpha , leu2, 3-112, his3-1, ura3-52, pgm2Delta ::HIS3, pgm1Delta ::KanR) (40) strains were a kind gift from Dr. J. M. François. FY833 (Mata, his3Delta 200, ura3-52, leu2Delta 1, lys2Delta 202, trp1Delta 63, GAL2+) was a kind gift from Dr. M. Ghislain. Expression plasmid pVRH3 (a gift from Dr. S. Verjovski-Almeida) is a multicopy plasmid with the strong promoter and the 3'-untranslated region from the yeast PMA1 gene. PGM2 open reading frame was amplified from yeast genomic DNA by PCR using Pfu DNA polymerase from 50 bp upstream of the start codon to 23 bp downstream of the stop codon using the primers PGM2f (5'-agggagctcaggatcaaccaatatttctcagt-3') and PGM2r (5'-cgggatccaagccattagtaaatcattcgt-3'). PCR products were cloned in pBlueScript II SK+ using BamHI and SacI restriction enzyme sites introduced in the primers. The open reading frame was subcloned to pVRH3 expression vector using the same restriction enzyme sites (pPGM2). Yeast strains JF-pVRH3 and JF-PGM2 were constructed by transforming JF291 strain with plasmids pVRH3 and pPGM2, respectively, by the lithium acetate method (41).

Culture Medium-- E. coli strain XL1-Blue was grown in LB medium with or without 100 µg/ml ampicillin. Yeast strains were selected in minimal medium (0.67% yeast nitrogen base) containing 2% of either glucose or galactose and the appropriate amino acids. After selection, they were grown in rich YP medium containing 2% Bacto-peptone, 1% yeast extract, and either 2% sugars or 3% glycerol or ethanol. When solid medium was used, 2% agar was added. Lithium (LiCl) and magnesium (MgCl2) were added at the indicated concentrations.

Growth Tests-- Yeast cells were grown in liquid YPGal or YPGly medium to mid-log phase and diluted to A600 of 0.3, 0.03, and 0.003. 4 µl of each dilution were dropped in the appropriate medium. Plates were incubated at 30 °C for 2-4 days and photographed.

Extraction and Analysis of Yeast Metabolites-- Extraction of metabolites was done based on previously published protocols (42, 43). Yeast cells were grown as described, and aliquots of the culture were collected by rapid vacuum filtration in 0.45-µm Millipore filters. Cells were immediately transferred to 75% ethanol at 80 °C and incubated for 3 min. Samples were lyophilized, resuspended in 10 mM Tris-HCl, pH 7.5, and 1 mM EDTA, and centrifuged at 20,000 × g for 20 min at 4 °C, and the supernatant was analyzed by high pH anion exchange chromatography (HPAEC), using a Dionex DX 500 system equipped with a CarboPac PA10 column (4 × 250 mm). Samples (25 µl) were applied and eluted with a stepwise gradient of 125 mM sodium acetate in 100 mM NaOH for 15 min and then with 180 mM sodium acetate in 100 mM NaOH for 30 min in a flow rate of 1 ml/min.

For UDP-glucose measurements, metabolites were extracted as described above except that lyophilized material was resuspended in 1 ml of buffer containing 200 mM Tris, pH 8.9, 5 mM MgCl2, and 2 mM NAD+ (44). UDP-glucose content was determined by measuring A340 before and after addition of 0.01 unit of UDP-glucose dehydrogenase (Sigma) into the solution.

Cell-free Extract of Soluble Proteins from Yeast-- Yeast cells were grown in YPGal to mid-log phase. Cells were harvested and washed with an equal amount of distilled water and resuspended in lysis buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 5 mM beta -mercaptoethanol, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 2 µg/ml chymostatin). An equal amount of 0.5 mm acid-washed glass beads was added, and cells were lysed by vortexing six times for 1 min followed by a 1-min rest on ice. The lysate was collected to another tube and centrifuged at 20,000 × g for 20 min at 4 °C. The supernatant was collected, and glycerol was added to a final concentration of 20% (v/v). Aliquots were frozen in liquid nitrogen and then stored at -70 °C until use.

Cell-free Extract of Soluble Proteins from HEK293 Cells-- HEK293 cells (kindly provided by Dr. H. Wolosker) were cultivated in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum in 100-mm dishes at 37 °C with an atmosphere of 5% CO2. Cells were scrapped from dishes and collected by centrifugation at 12,000 × g at 4 °C for 1 min. The cell pellet was resuspended in buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaF, 1 mM EDTA, 600 mM sucrose, 5 mM beta -mercaptoethanol, and 2 µg/ml amounts of each protease inhibitor (aprotinin, leupeptin, chymostatin, and pepstatin). Cells were lysed in a Teflon pestle tissue homogenizer with 30 strokes on ice. The cell lysate was centrifuged at 20,000 × g at 4 °C for 20 min. Supernatant was collected, and glycerol was added to a final concentration of 20%. Aliquots were frozen at -70 °C until use.

Biochemical Methods-- PGM and GK activity were measured using coupled systems basically as described (45). Briefly, for measuring PGM activity, an extract of yeast soluble proteins (50 µg/ml) was incubated at 23 °C in a buffer containing 50 mM Tris-HCl, pH 7.5, 0.5 mM NAD+, 2 units/ml recombinant Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase, and 1 mM MgCl2 unless otherwise stated. The reaction was started by the addition of 4 mM glucose 1-phosphate, and NADH formation was monitored by following A340. PGM activity is represented as micromoles of Glc-6-P mg-1 min-1 (units/mg) or as percentage of the control point.

GK activity was measured by incubating an extract of yeast soluble protein (50 µg/ml) at 23 °C in a buffer containing 20 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 50 mM NaCl, 1 mM phosphoenolpyruvate, 1 mM dithiothreitol, 0.3 mM NADH, 1 mM ATP, and 7/10 units/ml pyruvate kinase/lactate dehydrogenase enzyme mixture. The reaction was started by the addition of 5 mM galactose, and NADH oxidation was monitored by following A340. GK activity is represented as µmol of Gal-1-P mg-1 min-1 (units/mg).

Protein concentration was determined by the Lowry method (46) using bovine serum albumin as standard. Trehalose and glycogen content were determined as described (47).

Fermentation Rate-- The fermentation rate was measured by monitoring ethanol production (45, 48). Yeast cells were grown to mid-log phase (A600 ~ 1.0) in YPGal or YPD medium and washed twice with water. Cells were resuspended in water and incubated for another 4 h at 30 °C with shaking. Fermentation was measured by incubating cells in buffer containing 50 mM glucose or 50 mM galactose in 5 mM MES-triethanolamine, pH 6.0.

Northern Blotting-- Yeast cells were grown to mid-log phase (A600 ~ 1.0) in YPGal, and 15 mM LiCl was added to the culture. Aliquots of the culture were collected at different time intervals. 10 µg of total cellular RNA (49) were separated in a formaldehyde-denaturing agarose gel (50) and transferred to Immobilon-N+ membrane, UV-cross-linked, and pre-hybridized with 6× SSPE, 5× Denhardt's, 0.1% SDS at 65 °C. Probes for PGM1, PGM2, and ACT1 genes were amplified from yeast genomic DNA by PCR using Taq DNA polymerase. Primers PGM1fc (5'-ataggcgtaaccctgaaaagg-3') and PGM1r (5'-cgggatccggaaaattagtgcttgttcaagac-3') were used to produce PGM1 probe (+1226 bp from the ATG to +32 bp downstream the stop codon). To produce PGM2 probe (+1222 bp from ATG to +23 bp from the stop codon), primers PGM2fc (5'-aacaagcatcatccggagaac-3') and PGM2r were used in the reaction. ACT1 probe was amplified as described previously (28). Probes were labeled using random primers. Hybridization occurred at 65 °C in 6× SSPE, 0.5% SDS, and >1 × 106 cpm/ml of the corresponding probe for at least 16 h. Membranes were washed one time with 0.2× SSPE, 0.1% SDS at room temperature for 10 min and two times at 65 °C for 15 min and exposed to Kodak X-AR5 autoradiographic film with intensifying screen for 2-10 days at -70 °C.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Lithium Is More Toxic to Yeast Cells Growing in Galactose Than in Other Carbon Sources-- We have observed previously (28) that lithium is more toxic to yeast cells growing in galactose medium compared with yeast growing in glucose. This result was intriguing since it was observed that ENA1 gene, which expression is one of the main determinants of lithium and sodium tolerance in yeast (51), is under glucose repression presenting a 10-fold higher level in galactose compared with glucose (52). In this work, we have tested other repressing and non-repressing carbon sources for lithium tolerance. Wild type yeast strain JF291 was grown in rich (YP) medium containing several different carbon sources in the presence or absence of 30 mM LiCl. Fig. 2 shows that yeast cells can grow well in the presence of 30 mM LiCl in all carbon sources tested (see Fig. 2 legend), but not in galactose. This result indicates that the toxicity is galactose-specific and not related to glucose repression.


View larger version (66K):
[in this window]
[in a new window]
  		Fig. 2.   Galactose-specific lithium toxicity. Wild type yeast strain JF291 was grown in liquid YPGly to mid-log phase and then diluted to A600 of 0.3, 0.03, and 0.003. 4 µl of each dilution were plated in YP medium with different carbon sources in the absence or presence of 30 mM LiCl. Plates were incubated at 30 °C for 2-5 days and photographed. The carbon sources used were: YPGal, 2% galactose; YPD, 2% glucose; YPFru, 2% fructose; YPSuc, 2% sucrose; YPGly, 3% glycerol. We have also tested YP medium containing 3% ethanol, 2% raffinose, 2% maltose, and 2% lactose, and no growth inhibition by 30 mM LiCl was recorded (data not shown). The same results were obtained for yeast strain FY833.

It had been shown that Hal2p/Met22p, the yeast BPNase, is one of the main targets of lithium in yeast growing in glucose medium (24, 53). In the absence of methionine, inhibition of Hal2p by lithium causes an accumulation of its substrate 3',5'-bisphosphoadenosine (53), which is toxic to cells (54). High 3',5'-bisphosphoadenosine concentrations inhibit RNase MRP and Xrn1p, thus inhibiting RNA processing (54). All these effects are reverted by addition of methionine in culture medium (54, 55). We observed that addition of methionine did not confer any detectable protection against lithium toxicity in galactose medium (data not shown). Thus, the galactose-specific lithium toxicity is not related to the inhibition of Hal2p/Met22p.

Lithium Inhibits Yeast Phosphoglucomutase Activity-- Galactose is metabolized in yeast by the Leloir pathway (29, 30), which converts galactose to Glc-1-P that is further converted to Glc-6-P to enter glycolysis (Fig. 1). In Fig. 3A, we show an HPAEC analysis of some of the intermediate metabolites of this pathway (Gal-1-P, Glc-1-P, and Glc-6-P) extracted from a wild type yeast cell growing in YPGal medium in the absence or presence of 30 mM LiCl. Lithium induces a large accumulation of Gal-1-P and Glc-1-P and a small decrease in Glc-6-P levels. No change in the steady state levels of trehalose 6-phosphate (Tre-6-P) and mannose 6-phosphate (Man-6-P) was detected. This result could be explained by inhibition of PGM, the enzyme that converts Glc-1-P to Glc-6-P. To test this hypothesis, we assayed the yeast PGM activity in vitro in the presence of increasing lithium concentrations. We show (Fig. 3B) that lithium inhibits with high affinity (IC50 of 200 µM in the presence of 1 mM MgCl2) the yeast PGM activity and that sodium and potassium inhibit only at very high concentrations (IC50 > 50 mM). This inhibition is not caused by Cl- anion since other Li+, Na+, and K+ salts have the same effect (data not shown).


View larger version (18K):
[in this window]
[in a new window]
  		Fig. 3.   Lithium inhibits yeast PGM activity. A, HPAEC analysis of metabolites of the Leloir pathway. Wild type yeast strain JF291 was grown in liquid YPGal medium at 30 °C to A600 ~ 1.0. LiCl was added to half of the culture to a final concentration of 15 mM and incubated for 3.5 h. Yeast metabolites were extracted and analyzed as described under "Experimental Procedures." Analysis of cells treated (upper panel) or not (lower panel) with lithium are presented. The identified metabolites galactose-1-P (Gal-1-P), glucose-1-P (Glc-1-P), trehalose-6-P (Tre-6-P), glucose-6-P (Glc-6-P), and mannose-6-P (Man-6-P) are indicated in the figure. B, PGM activity. A cell-free extract of yeast soluble proteins was assayed for PGM activity as described under "Experimental Procedures" in the presence of increasing concentrations of LiCl (), NaCl (), and KCl (black-triangle). Results represent mean values of three independent determinations. 100% activity corresponds to 0.244 units/mg.

Conversion of Glc-1-P to Glc-6-P is an essential step for glycolysis when yeast cells are growing with galactose as the sole carbon source (see Fig. 1). However, when growing with glucose, glycolysis does not depend on PGM activity. We measured the fermentation rate in both carbon sources. In glucose, the fermentation rate was 66.4 ± 16.0 and 63.0 ± 15.4 (nmol ethanol/min/mg dry weight) in the absence or presence of 100 mM LiCl, respectively. In galactose, the fermentation rate was 44.6 ± 3.6 and 4.3 ± 2.2 (nmol ethanol/min/mg dry weight) in the absence or presence of 15 mM LiCl, respectively. Galactose fermentation is very sensitive to lithium, whereas glucose fermentation is not inhibited. These results indicate that lithium inhibits both in vitro and in vivo the yeast PGM activity.

Increased Magnesium Concentrations Protect PGM Activity Both in Vitro and in Vivo-- It had been shown that lithium inhibits rabbit PGM by displacing magnesium, a cofactor of the enzyme, from its binding site (22, 23). As we can see in Fig. 4A, increasing the magnesium concentration protects PGM activity from lithium inhibition. Fig. 4B shows the Dixon plot of the same experiment that presents a pattern typical of a competitive inhibition. From this plot we can also calculate an apparent Ki for lithium of ~60 µM. Neither the substrate Glc-1-P nor glucose 1,6-bisphosphate, an activator of the enzyme, protects PGM activity from lithium inhibition (data not shown). It was shown that high potassium concentration could protect yeast BPNase activity (Hal2p) from lithium inhibition (24). However, addition of 100 mM KCl in the assay medium had no effect on PGM activity in the presence of lithium (data not shown).


View larger version (21K):
[in this window]
[in a new window]
  		Fig. 4.   Magnesium reverts inhibition of PGM activity by lithium both in vitro and in vivo. A, PGM activity. A cell-free extract of yeast soluble proteins was assayed for PGM activity as described under "Experimental Procedures" in the presence of 0.1 (black-square), 1.0 (black-triangle), and 10 mM () MgCl2. 100% activity corresponds to 0.104, 0.235, and 0.247 units/mg for 0.1, 1.0, and 10 mM MgCl2, respectively. This result is a mean of four independent determinations. B, Dixon's plot of the experiment presented in panel A showing a typical pattern of a competitive inhibition with magnesium. Symbols are the same ones used in panel A. C, yeast strain FY833 was grown in liquid YPGal to mid-log phase and then diluted to A600 of 0.3, 0.03, and 0.003. 4 µl of each dilution were plated in YPGal and YPGal plus 100 mM MgCl2 in the absence or presence of 15 mM LiCl. Plates were incubated at 30 °C for 3 days, and growth was recorded. Similar results were obtained for yeast strain JF291. D, effect of magnesium on metabolites of the Leloir pathway. Two cultures of the wild type yeast strain FY833 were grown in liquid YPGal medium without (upper panel) or with (lower panel) 100 mM MgCl2 at 30 °C to A600 ~ 1.0. LiCl was added in both cultures to the final concentration of 10 mM and were incubated for 3.5 h. Yeast metabolites were extracted and analyzed as described under "Experimental Procedures." The identified metabolites are indicated.

We also show that the addition of 100 mM MgCl2 to YPGal medium protects yeast cells from lithium toxicity (Fig. 4C). In order to determine if the reversion of lithium toxicity conferred by magnesium is a consequence of a protection of the PGM activity in vivo, we have analyzed the intermediate metabolites of the Leloir pathway. Metabolites from wild type yeast cells grown in YPGal medium with 10 mM lithium in the presence or absence of 100 mM MgCl2 were extracted and analyzed by HPAEC. Fig. 4D shows that addition of magnesium prevents the accumulation of Gal-1-P and Glc-1-P induced by lithium. These results clearly demonstrate that adding magnesium to the growth medium is able to prevent lithium inhibition of PGM activity in vivo.

Inhibition of PGM Activity Is a Major Cause of Lithium Toxicity to Yeast Growing in Galactose Medium-- Boles and co-workers (36) had shown previously that PGM activity is essential for yeast growth in galactose medium, but not in glucose or glycerol plus ethanol. We have extended this study to other carbon sources. Fig. 5A shows that the pgm1pgm2 mutant grows in all carbon sources tested except galactose. This pattern is identical to that observed for wild type strains growing in the presence of 30 mM LiCl (Fig. 2). Furthermore, we have analyzed the intermediate metabolites of galactose metabolism in the pgm1pgm2 double mutant strain incubated in YPGal medium and observed higher steady-state levels of Gal-1-P and Glc-1-P compared with a wild type strain (Fig. 5B). This result is similar to that observed for yeast grown in the presence of lithium (Fig. 3A). Unlike from what we had observed with lithium, magnesium could not protect this mutant from the growth defect in galactose (data not shown). These results indicate that inhibition of PGM activity is a major cause of lithium toxicity to yeast in galactose.


View larger version (29K):
[in this window]
[in a new window]
  		Fig. 5.   pgm1pgm2 double mutant has the same phenotype of a wild type strain growing in the presence of lithium. A, yeast strain JF645 (pgm1pgm2) was grown in liquid YPGly to mid-log phase and then diluted to A600 of 0.3, 0.03, and 0.003. 4 µl of each dilution were plated in YP medium without LiCl with different carbon sources as described in Fig. 2. Plates were incubated at 30 °C for 2-5 days and photographed. Growth was recorded in all carbon sources tested, except galactose. B, yeast strains JF291 (lower panel) and JF645 (upper panel) were grown in liquid YPGly medium at 30 °C to A600 ~ 1.0. Cells were collected by centrifugation, resuspended in YPGal medium, and incubated for 5 h, after which metabolites were extracted and analyzed as described under "Experimental Procedures." The identified metabolites are indicated.

Lithium Stress Induces PGM Activity-- Yeast PGM expression is modulated by many different external stimuli such as heat shock, DNA damage (56), oxidative stress (57), and carbon source (35, 39, 58). To address whether PGM expression is induced by lithium stress, we have analyzed the mRNA levels of the two yeast genes (PGM1 and PGM2) encoding PGM by Northern blotting. Fig. 6 shows that expression of the PGM2 gene is highly induced within 20 and 40 min of lithium addition. The level of PGM2 mRNA decreases after 60 min, but is still higher than that of the control condition. No signal of PGM1 mRNA could be detected under the same experimental conditions used for PGM2 (data not shown).


View larger version (48K):
[in this window]
[in a new window]
  		Fig. 6.   PGM2 gene is induced by lithium stress. Wild type yeast strain FY833 was grown in YPGal medium to mid-log phase. 15 mM LiCl was added to the medium, and aliquots of the culture were taken at the indicated times for total RNA purification. PGM2 and ACT1 mRNAs were analyzed by Northern blot using 10 µg of total RNA/lane. Blots were exposed to autoradiographic film for ~48 h at -80 °C with intensifying screen. The same filter was used for both analysis. This is a representative result of two independent experiments.

We also measured if the increased transcription was correlated with an increased PGM activity in yeast cells. We prepared a cell-free extract of soluble proteins from yeast grown in YPGal medium treated or not with 15 mM LiCl for 3 h. Table I shows that yeast cells stressed with lithium present a higher PGM specific activity compared with non-stressed yeast cells. This effect is not a general induction of the pathway since we could not observe any difference in the specific activity of GK (Table I). These results show that PGM activity is induced by lithium stress due to an induction of PGM2 gene.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Lithium stress induces PGM activity in yeast
GK and PGM activities from yeast cells incubated in YPGal medium with or without 15 mM LiCl for 3 h were assayed as described under "Experimental Procedures." Results are represented as mean ± S.D. Numbers in parentheses indicate the number of experiments.

Overexpression of PGM2 Gene Confers Lithium Tolerance-- We have cloned the complete open reading frame of the PGM2 gene in a multicopy expression vector under the control of the strong PMA1 gene promoter. Yeast strain overexpressing PGM2 gene (JF-PGM2) presents a 4.0-fold increase in PGM activity (1.07 ± 0.16 units/mg) in comparison with the control strain (JF-pVRH3) transformed with the empty plasmid (0.27 ± 0.06 units/mg). Fig. 7A shows that JF-PGM2 strain with higher PGM activity is more tolerant to lithium stress in galactose medium than the control strain JF-pVRH3. Additionally, overexpression of PGM2 gene partially reverts the accumulation of Gal-1-P and Glc-1-P induced by lithium (Fig. 7B). These results support the idea that inhibition of PGM activity is a major cause of lithium toxicity for yeast growing in galactose medium.


View larger version (28K):
[in this window]
[in a new window]
  		Fig. 7.   Overexpression of PGM2 protects yeast cells from lithium effects. A, yeast strain JF-pVRH3 (control) and JF-PGM2 (PGM2) were grown in liquid YPGal medium to mid-log phase and then diluted to A600 of 0.3, 0.03, and 0.003. 4 µl of each dilution were plated in YPGal medium with the indicated concentrations of LiCl. Plates were incubated at 30 °C for 3 days and photographed. B, yeast strains JF-pVRH3 (upper panel) and JF-PGM2 (lower panel) were grown in liquid YPGal medium at 30 °C to A600 ~ 1.0. LiCl was added to the final concentration of 10 mM. Cultures were incubated for 3.5 h, and metabolites were extracted and analyzed as described under "Experimental Procedures." The identified metabolites are indicated.

Lithium Also Inhibits PGM Activity of Yeast Cells Growing in Glucose Medium-- Low lithium concentrations inhibit yeast PGM activity (Fig. 3). However, concentrations as high as 100 mM do not prevent yeast growth in glucose (28). Deletion of both PGM1 and PGM2 genes is not lethal in glucose, but reduces the levels of UDP-glucose (UDP-Glc) (40). We measured the steady-state level of UDP-Glc of glucose-growing cells treated or not with lithium (Fig. 8A). After addition of 100 mM LiCl, UDP-Glc levels of a wild type yeast strain (JF291) decrease rapidly. After only 20 min, the UDP-Glc content is reduced (~ 60% reduction) to levels comparable to those observed for the pgm1pgm2 -deleted mutant (JF645). This decrease in UDP-Glc level is not observed in the strain overexpressing the PGM2 gene (JF-PGM2).


View larger version (28K):
[in this window]
[in a new window]
  		Fig. 8.   Effects of PGM inhibition by lithium on glucose grown cells. A, UDP-Glc levels. Yeast strains JF645 (pgm1pgm2), JF291 (WT), and JF-PGM2 (PGM2) were grown in minimal medium containing glucose as the sole carbon source (SD medium) at 30 °C to A600 ~ 2.0. LiCl (100 mM) was added to the culture, and metabolites were extracted from cells incubated for 0 (white bars), 10 (dotted bars), 20 (dashed bars), and 30 min (gray bars); and UDP-Glc was measured as described under "Experimental Procedures." Results are mean ± S.D. of three experiments. B, trehalose synthesis. Yeast strains were grown in YPD medium in the absence (white bars) or presence (black bars) of 50 mM LiCl at 30 °C to A600 ~ 1.0. Cultures were further submitted to a heat-shock treatment (40 °C) for 60 min to induce trehalose synthesis. Cells were collected by filtration, and trehalose was measured. Results are mean ± S.D. of four experiments. C, glycogen content. Yeast strains were grown in SD medium in the absence (white bars) or presence (black bars) of 50 mM LiCl at 30 °C to A600 ~ 2.0. Cells were collected by filtration, and glycogen was measured as described under "Experimental Procedures." Results are mean ± S.D. of six experiments. *, p < 0.01, t test. **, p < 0.001, t test.

Reduction of UDP-Glc levels induced by deletion of PGM1/PGM2 genes (36, 40) or by reduction of UDP-glucose pyrophosphorylase activity (40) cause a decrease on trehalose and glycogen content. We measured glycogen and trehalose content after lithium treatment in order to test whether the reduced UDP-Glc level was affecting these functions. Fig. 8B shows that trehalose synthesis induced by heat shock in the wild type strain JF291 is inhibited 30% by lithium treatment. Yeast strain JF645 (pgm1pgm2) accumulates trehalose in levels similar to the lithium-treated cells and overexpression of PGM2 protects from the lithium effect. Similar results were observed for glycogen synthesis (Fig. 8C), although the decrease (~15%) in glycogen content observed is smaller. These results show that PGM activity is also inhibited by lithium in glucose-grown yeast cells. It affects the UDP-Glc levels and its further metabolism to glycogen and trehalose.

Human PGM Is Also Inhibited by Lithium-- Lithium is widely used for the treatment of manic depressive disorder (1, 2). To investigate whether human PGM is also sensitive to lithium, we assayed PGM activity in a cell-free extract of soluble proteins from HEK293 cells. Fig. 9 shows that human PGM is also inhibited by lithium with an IC50 of ~1.5 mM in the presence of 1 mM MgCl2. This value is higher than that of the yeast PGM (IC50 = 200 µM), indicating that the affinity of human PGM for lithium is lower. However, this concentration is still therapeutically relevant (1, 3). Interestingly, this inhibition is also competitive with magnesium (Fig. 9), suggesting that the mechanism of lithium inhibition is similar in the yeast and human PGM.


View larger version (14K):
[in this window]
[in a new window]
  		Fig. 9.   Human PGM activity is also inhibited by lithium. PGM activity from a cell-free extract of HEK293 cells was assayed as described under "Experimental Procedures" in the presence of increasing lithium concentrations and 0.1 (black-square), 1.0 (black-triangle), and 10 () mM MgCl2. 100% activity corresponds to 0.087, 0.111, and 0.073 units/mg for 0.1, 1.0, and 10 mM MgCl2, respectively. This result is a mean of three independent experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this work we have characterized the galactose-specific lithium toxicity observed in yeast. This toxicity is caused by the inhibition of yeast PGM activity by lithium. Analysis of the steady state levels of the Leloir pathway metabolites revealed a large accumulation of Gal-1-P and Glc-1-P and a small decrease in Glc-6-P levels when yeast cells are grown in galactose in the presence of lithium (Fig. 3A). This accumulation is the result of the in vivo inhibition of PGM activity by lithium. This result is corroborated by the metabolite analysis of the strain pgm1pgm2 deleted in both isoforms of PGM, which shows essentially the same result when incubated in medium with galactose as the sole carbon source (Fig. 5B). Inhibition of PGM activity is one of the major problems caused by lithium to yeast cells growing in galactose medium since overexpression of PGM2 gene (Fig. 7A) or protection of PGM activity by high magnesium concentrations (Fig. 4C) confer lithium tolerance. Lithium inhibits fermentation rates in galactose, but not in glucose. These results indicate that lithium toxicity in galactose is associated with the inhibition of fermentation.

Although lithium does not inhibit fermentation in glucose-grown cells, it inhibits phosphoglucomutase activity. The major consequence of this inhibition is a 60% reduction of the UDP-Glc levels (Fig. 8A). This value is comparable to that obtained in the pgm1pgm2 strain. In accordance with previous results obtained in a pgm1pgm2 strain (40), lithium does not reduce UDP-Glc levels completely, suggesting that yeast cells have other compensatory pathways for UDP-Glc synthesis. We further show that the effect of lithium on reduction of UDP-Glc levels is abolished by PGM2 overexpression (Fig. 8A).

UDP-Glc is an intermediary metabolite of various biosynthetic pathways including glycogen and trehalose biosynthesis, formation of cell wall beta -glucans, and protein glycosylation. Consequently, reduction in UDP-Glc levels by lithium could inhibit these pathways. We have measured the effect of lithium treatment on trehalose (Fig. 8B) and glycogen (Fig. 8C) biosynthesis. Our results are in accordance with previously published results showing that reduced UDP-Glc levels inhibit both processes (40). Trehalose synthesis induced by heat shock is inhibited 40% by lithium, although glycogen levels are reduced only 15%. The smaller effect observed in glycogen levels should be due to a compensatory regulation between glycogen degradation and biosynthesis. In fact, it has been shown that lithium activates the yeast glycogen synthase activity (56, 57).

Another physiological alteration due to PGM inhibition would be the N-glycosylation of proteins. UDP-Glc is the sugar donor for the formation of dolichol-P-glucose that is used to form the core oligosaccharide for protein N-glycosylation (58-63). In yeast the dolichol-P-glucose synthase is encoded by ALG5 (64). Deletion of ALG5 is not lethal but results on underglycosylation of invertase and carboxypeptidase Y (64, 65). Reduction of UDP-Glc levels by lithium would probably lead to hypoglucosylation of the core glycan. Protein N-glycosylation is important in many processes such as protein folding, sorting, transport, and degradation (66, 67). It was shown that reduction in UDP-Glc levels by diminishing the expression of UDP-glucose pyrophosphorylase leads to modifications on the cell wall composition, although no major modification on protein glycosylation was observed (68). In mammalian cells, where we and others (22, 23) have shown that phosphoglucomutase is also inhibited by lithium, UDP-Glc plays an important role in the calnexin-calreticulin cycle. It is the glucose donor for the UDP-Glc:glycoprotein glucosyltransferase, the enzyme that behaves as a sensor of glycoprotein conformation in endoplasmic reticulum (66, 67). Although the calnexin-calreticulin cycle is not present in yeast (66), lithium could affect this process in mammalian cells.

Lithium potently inhibits yeast PGM activity in vitro with an apparent Ki of ~60 µM (Fig. 4B). On the other hand, sodium and potassium are not good inhibitors of the enzyme (IC50 > 50 mM). Previous reports have shown that rabbit PGM (22, 23) is inhibited by lithium. Lithium binds with high affinity (Kd ~10 µM) to the magnesium-binding site of rabbit PGM, displacing the cofactor from the rabbit enzyme (22) and inhibiting its activity (23). In yeast, PGM inhibition by lithium is competitive with magnesium, suggesting that lithium is also displacing magnesium from the enzyme by competing for its binding site.

Other enzymes, such as IMPase (Ki = 0.26-1.0 mM) (6, 7), IPPase (Ki = 0.3 mM) (8), BPNase (Ki = 0.1-0.6 mM) (24-26), and FBPase (Ki = 0.3-0.8 mM) (20, 21) are also inhibited by lithium. All these enzymes share a structural motif, which includes a metal binding site, that confers lithium-binding capacity (27). We have searched for this motif in Pgm1p and Pgm2p sequences, and we could not detect its presence. This observation suggests that the lithium-binding site in PGM is structurally different from that of IMPases, IPPases, BPNase, and FBPase.

Lithium stress increases PGM-specific activity in yeast extracts (Table I). This increment is due to an induction of the PGM2 gene (Fig. 6). The PGM2 gene expression is regulated by a number of external stimuli, such as heat shock and DNA damage (69), oxidative stress (70), and carbon source (35, 39, 71). Recently, it was reported that both PGM1 and PGM2 genes are induced by NaCl stress (72, 73). At least some of these responses depend on the function of Msn2p/Msn4p transcription factors (69, 71) via the stress response element (STRE), whose sequence is AGGGG. There are five STRE sequences in the PGM2 gene promoter (-500 bp upstream from the ATG). Whether this induction caused by lithium is also dependent on the STRE sequences and Msn2p/Msn4p transcription factors remains to be determined.

We have shown previously that overexpression of the SIT4 gene, which encodes a Ser-Thr protein phosphatase, enhances lithium tolerance to yeast cells growing in galactose medium (28). Preliminary results show that PGM activity is not enhanced in the SIT4-overexpressing cells, suggesting that another mechanism is responsible for the tolerance of these cells. Studies on the involvement of the SIT4 in carbohydrate metabolism could also give an insight in the mechanism of lithium toxicity.

In humans, lithium is used in the treatment of manic-depressive disorder (1, 2). Interestingly, Parthasarathy and co-workers (74) had shown that brain IMPase, an enzyme also inhibited by lithium, is able to use Gal-1-P as substrate with the same efficiency as it uses D-1- and D-3-myo-inositol monophosphates (74). Here we present experimental evidence that the human PGM activity is inhibited by therapeutic relevant lithium concentrations. Together, these results suggest that galactose metabolism could be a target of lithium action in humans. However, it is important to note that PGM activity is involved in other important cellular processes such as protein glycosylation, polysaccharide biosynthesis, and glycogen metabolism. Lithium action in these processes should be investigated in the future.

    ACKNOWLEDGEMENTS

We thank Rogério Panizzutti and Dr. H. Wolosker (Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil) for providing HEK293 cultures, Dr. J. M. François (Institut National des Sciences Appliquées, Toulouse, France) for providing yeast strains, and Dr. L. M. Previato and Dr. J. O. Previato (UFRJ, Rio de Janeiro, Brazil) for making available the HPAEC equipment for metabolites measurement and for helpful discussions.

    FOOTNOTES

* This work was supported by grants from Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) (to M. M.-L. and A. G.); fellowships from Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq); and grants (to Dr. L. de Meis, UFRJ, Brazil) from Financiadora de Estudos e Projetos/Pronex, CNPq, and FAPERJ.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 55-21-2590-4548; Fax: 55-21-2270-8647; E-mail: montero@server.bioqmed.ufrj.br.

|| A Ph.D. student from Instituto de Microbiologia, UFRJ.

Published, JBC Papers in Press, August 10, 2001, DOI 10.1074/jbc.M101451200

    ABBREVIATIONS

The abbreviations used are: IMPase, inositol monophosphatase; PGM, phosphoglucomutase; GK, galactokinase; IPPase, inositol polyphosphate phosphatase; BPNase, 3'(2'),5'-bisphosphate nucleotidase; FBPase, fructose 1,6-bisphosphatase; Gal-1-P, galactose 1-phosphate; Glc-1-P, glucose 1-phosphate; Glc-6-P, glucose 6-phosphate; Tre-6-P, trehalose 6-phosphate; Man-6-P, mannose 6-phosphate; UDP-Glc, uridine 5'-diphosphoglucose; STRE, stress response element; HPAEC, high pH anion exchange chromatography; PCR, polymerase chain reaction; bp, base pair(s); MES, 4-morpholineethanesulfonic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Schou, M. (1997) Arch. Gen. Psychiatry 54, 9-13
2. Jope, R. S. (1999) Mol. Psychiatry 2, 117-128
3. Manji, H. K., Potter, W. Z., and Lenox, R. H. (1995) Arch. Gen. Psychiatry 52, 531-543
4. Williams, R. S. B., and Harwood, A. J. (2000) Trends Pharmacol. Sci. 21, 61-64
5. Berridge, M. J., Downes, C. P., and Hanley, M. R. (1989) Cell 59, 411-419
6. Hallcher, L. M., and Sherman, W. R. (1980) J. Biol. Chem. 255, 10896-10901
7. Gee, N. S., Ragan, C. I., Watling, K. J., Aspley, S., Jackson, R. G., Reid, G. G., Gani, D., and Shute, J. K. (1988) Biochem. J. 249, 883-889
8. Inhorn, R. C., and Majerus, P. W. (1988) J. Biol. Chem. 263, 14559-14565
9. Klein, P. S., and Melton, D. A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 8455-8459
10. Stambolic, V., Ruel, L., and Woodgett, J. R. (1996) Curr. Biol. 6, 1664-1668
11. Wodarz, A., and Nusse, R. (1998) Annu. Rev. Cell Dev. Biol. 14, 59-88
12. Kao, K. R., Masui, Y., and Elinson, R. P. (1986) Nature 322, 371-373
13. Livingston, B. T., and Wilt, F. H. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 3669-3673
14. Peters, D. J. M., Van Lookeren-Campagne, M. M., Van Haastert, P. J. M., Spek, W., and Schaap, P. (1989) J. Cell Sci. 93, 205-210
15. Jerka-Dziadosz, M., and Frankel, J. (1995) Dev. Biol. 171, 497-506
16. Harwood, A. J., Plyte, S. E., Woodgett, J., Strutt, H., and Kay, R. R. (1995) Cell 80, 139-148
17. He, X., Saint-Jeannet, J. P., Woodgett, J. R., Varmus, H. E., and Dawid, I. B. (1995) Nature 374, 617-622
18. Dominguez, I., Itoh, K., and Sokol, S. Y. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8498-8502
19. Pierce, S. B., and Kimelman, D. (1995) Development 121, 755-765
20. Nakashima, K., and Tuboi, S. (1976) J. Biol. Chem. 251, 4315-4321
21. Marcus, F., and Hosey, M. M. (1980) J. Biol. Chem. 255, 2481-2486
22. Ray, W. J., Jr., Szymanki, E. S., and Ng, L. (1978) Biochim. Biophys. Acta 522, 434-442
23. Rhyu, G. I., Ray, W. J., Jr., and Markley, J. L. (1984) Biochemistry 23, 252-260
24. Murguía, J. R., Bellés, J. M., and Serrano, R. (1995) Science 267, 232-234
25. Spiegelberg, B. D., Xiong, J. P., Smith, J. J., Gu, R. F., and York, J. D. (1999) J. Biol. Chem. 274, 13619-13628
26. Yenush, L., Bellés, J. M., López-Coronado, J. M., Gil-Mascarell, R., Serrano, R., and Rodríguez, P. L. (2000) FEBS Lett. 467, 321-325
27. York, J. D., Ponder, J. W., and Majerus, P. W. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 5149-5153
28. Masuda, C. A., Ramírez, J., Peña, A., and Montero-Lomelí, M. (2000) J. Biol. Chem. 275, 30957-30961
29. Kosterlitz, F. W. (1943) Biochem. J. 37, 322
30. Leloir, L. F. (1951) Arch. Biochem. 33, 186-190
31. Kalckar, H. M. (1957) Science 125, 105-108
32. Robichon-Szulmajster, H. (1958) Science 127, 28-29
33. Douglas, H. C., and Hawthorne, D. C. (1964) Genetics 49, 837-844
34. Douglas, H. C. (1961) Biochim. Biophys. Acta 52, 209-211
35. Oh, D., and Hopper, J. E. (1990) Mol. Cell. Biol. 10, 1415-1422
36. Boles, E., Liebetrau, W., Hofmann, M., and Zimmermann, F. K. (1994) Eur. J. Biochem. 220, 83-96
37. Douglas, H. C., and Condie, F. (1954) J. Bacteriol. 68, 662-670
38. Johnston, M. (1987) Microbiol. Rev. 51, 458-476
39. Fu, L., Bounelis, P., Dey, N., Browne, B. L., Marchase, R. B., and Bedwell, D. M. (1995) J. Bacteriol. 177, 3087-3094
40. Daran, J. M., Bell, W., and François, J. (1997) FEMS Microbiol. Lett. 153, 89-96
41. Ito, H., Fukuda, Y., Murata, K., and Kimura, A. (1983) J. Bacteriol. 153, 163-168
42. Entian, K.-D., Zimmermann, F. K., and Scheel, I. (1977) Mol. Gen. Genet. 156, 99-105
43. Gonzalez, B., François, J., and Renaud, M. (1997) Yeast 13, 1347-1356
44. Koo, S. J., Neal, J. C., and DiTomaso, J. M. (1996) Plant Physiol. 112, 1383-1389
45. Bergmeyer, H. U. (1986) Methods in Enzymatic Analysis , 3rd Ed. , Verlag Chemie, Weinheim, Germany
46. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275
47. Parrou, J. L., and François, J. (1997) Anal. Biochem. 248, 186-188
48. Ramírez, J., Ramírez, O., Saldaña, C., Coria, R., and Peña, A. (1998) J. Bacteriol. 180, 5860-5865
49. Schmitt, M. E., Brown, T. A., and Trumpower, B. L. (1990) Nucleic Acids Res. 18, 3091-3092
50. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
51. Serrano, R. (1996) Int. Rev. Cytol. 165, 1-48
52. Alepuz, P. M., Cunningham, K. W., and Estruch, F. (1997) Mol. Microbiol. 26, 91-98
53. Murguía, J. R., Bellés, J. M., and Serrano, R. (1996) J. Biol. Chem. 271, 29029-29033
54. Dicht, B., Stevens, A., and Tollervey, D. (1997) EMBO J. 16, 7184-7195
55. Gläser, H. U., Thomas, D., Gaxiola, R., Montrichard, F., Surdin-Kerjan, Y., and Serrano, R. (1993) EMBO J. 12, 3105-3110
56. Peng, Z-Y., Trumbly, R. J., and Reinmann, E. M. (1990) J. Biol. Chem. 265, 13871-13877
57. Posas, F., Clotet, J., and Ariño, J. (1991) FEBS Lett. 279, 341-345
58. Behrens, N. H., and Leloir, L. F. (1970) Proc. Natl. Acad. Sci. U. S. A. 66, 153-159
59. Behrens, N. H., Parodi, A. J., and Leloir, L. F. (1971) Proc. Natl. Acad. Sci. U. S. A. 68, 2857-2860
60. Parodi, A. J., Behrens, N. H., Leloir, L. F., and Carminatti, H. (1972) Proc. Natl. Acad. Sci. U. S. A. 69, 3268-3272
61. Parodi, A. J. (1977) Eur. J. Biochem. 75, 171-180
62. Lehle, L. (1980) Eur. J. Biochem. 109, 589-601
63. Kornfeld, R., and Kornfeld, S. (1985) Annu. Rev. Biochem. 54, 631-664
64. Heesen, S., Lehle, L., Weissmann, A., and Aebi, M. (1994) Eur. J. Biochem. 224, 71-79
65. Huffaker, T. C., and Robbins, P. W. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 7466-7470
66. Parodi, A. J. (2000) Annu. Rev. Biochem. 69, 69-93
67. Helenius, A., and Aebi, M. (2001) Science 291, 2364-2369
68. Daran, J. M., Dallies, N., Thines-Sempoux, D., Paquet, V., and François, J. (1995) Eur. J. Biochem. 233, 520-530
69. Treger, J. M., Schmitt, A. P., Simon, J. R., and McEntee, K. (1998) J. Biol. Chem. 273, 26875-26879
70. Godon, C., Lagniel, G., Lee, J., Buhler, J. M., Kieffer, S., Perrot, M., Boucherie, H., Toledano, M. B., and Labarre, J. (1998) J. Biol. Chem. 273, 22480-22489
71. Boy-Marcotte, E., Perrot, M., Busserreau, F., Boucherie, H., and Jacquet, M. (1998) J. Bacteriol. 180, 1044-1052
72. Posas, F., Chambers, J. R., Heyman, J. A., Hoeffler, J. P., Nadal, E., and Ariño, J. (2000) J. Biol. Chem. 275, 17249-17255
73. Yale, J., and Bohnert, H. J. (2001) J. Biol. Chem. 276, 15996-16007
74. Parthasarathy, R., Parthasarathy, L., and Vadnal, R. (1997) Brain Res. 778, 99-106

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 MicrobiologyHome page
J. van den Brink, M. Akeroyd, R. van der Hoeven, J. T. Pronk, J. H. de Winde, and P. Daran-Lapujade
Energetic limits to metabolic flexibility: responses of Saccharomyces cerevisiae to glucose-galactose transitions
Microbiology, April 1, 2009; 155(4): 1340 - 1350.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
G. McColl, D. W. Killilea, A. E. Hubbard, M. C. Vantipalli, S. Melov, and G. J. Lithgow
Pharmacogenetic Analysis of Lithium-induced Delayed Aging in Caenorhabditis elegans
J. Biol. Chem., January 4, 2008; 283(1): 350 - 357.
[Abstract] [Full Text] [PDF]

Home page
 Plant Physiol.Home page
E. Peiter, J. Sun, A. B. Heckmann, M. Venkateshwaran, B. K. Riely, M. S. Otegui, A. Edwards, G. Freshour, M. G. Hahn, D. R. Cook, et al.
The Medicago truncatula DMI1 Protein Modulates Cytosolic Calcium Signaling
Plant Physiology, September 1, 2007; 145(1): 192 - 203.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
Y. Jin, S. Weining, and E. Nevo
A MAPK gene from Dead Sea fungus confers stress tolerance to lithium salt and freezing-thawing: Prospects for saline agriculture
PNAS, December 27, 2005; 102(52): 18992 - 18997.
[Abstract] [Full Text] [PDF]

Home page
 Appl. Environ. Microbiol.Home page
C. Bro, S. Knudsen, B. Regenberg, L. Olsson, and J. Nielsen
Improvement of Galactose Uptake in Saccharomyces cerevisiae through Overexpression of Phosphoglucomutase: Example of Transcript Analysis as a Tool in Inverse Metabolic Engineering
Appl. Envir. Microbiol., November 1, 2005; 71(11): 6465 - 6472.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
P. Csutora, A. Strassz, F. Boldizsar, P. Nemeth, K. Sipos, D. P. Aiello, D. M. Bedwell, and A. Miseta
Inhibition of phosphoglucomutase activity by lithium alters cellular calcium homeostasis and signaling in Saccharomyces cerevisiae
Am J Physiol Cell Physiol, July 1, 2005; 289(1): C58 - C67.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
B. D. Spiegelberg, J. dela Cruz, T.-H. Law, and J. D. York
Alteration of Lithium Pharmacology through Manipulation of Phosphoadenosine Phosphate Metabolism
J. Biol. Chem., February 18, 2005; 280(7): 5400 - 5405.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Interv.Home page
J. A. Quiroz, T. D. Gould, and H. K. Manji
MOLECULAR EFFECTS of lithium
Mol. Interv., October 1, 2004; 4(5): 259 - 272.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
C. Bro, B. Regenberg, G. Lagniel, J. Labarre, M. Montero-Lomeli, and J. Nielsen
Transcriptional, Proteomic, and Metabolic Responses to Lithium in Galactose-grown Yeast Cells
J. Biol. Chem., August 22, 2003; 278(34): 32141 - 32149.
[Abstract] [Full Text] [PDF]

Home page
 Appl. Environ. Microbiol.Home page
K.-R. Chung
Involvement of Calcium/Calmodulin Signaling in Cercosporin Toxin Biosynthesis by Cercosporanicotianae
Appl. Envir. Microbiol., February 1, 2003; 69(2): 1187 - 1196.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
276/41/37794    most recent
M101451200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Masuda, C. A.
Right arrow Articles by Montero-Lomelí, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Masuda, C. A.
Right arrow Articles by Montero-Lomelí, M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2001 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement