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Published as doi: 10.1096/fj.07-104851.
 (The FASEB Journal. 2008;22:2206-2213.)
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The glucose-6-phosphate transporter is a phosphate-linked antiporter deficient in glycogen storage disease type Ib and Ic

Shih-Yin Chen*, Chi-Jiunn Pan*, Krishnamachary Nandigama{dagger}, Brian C. Mansfield*, Suresh V. Ambudkar{dagger} and Janice Y. Chou*,1

* Section on Cellular Differentiation, Heritable Disorders Branch, National Institute of Child Health and Human Development and

{dagger} Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA

1Correspondence: National Institutes of Health, Bldg. 10, Rm. 9D42, 10 Center Dr., Bethesda, MD 20892-1830, USA. E-mail: chouja{at}mail.nih.gov


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Glycogen storage disease type Ib (GSD-Ib) is caused by deficiencies in the glucose-6-phosphate (G6P) transporter (G6PT) that have been well characterized. Interestingly, deleterious mutations in the G6PT gene were identified in clinical cases of GSD type Ic (GSD-Ic) proposed to be deficient in an inorganic phosphate (Pi) transporter. We hypothesized that G6PT is both the G6P and Pi transporter. Using reconstituted proteoliposomes we show that both G6P and Pi are efficiently taken up into Pi-loaded G6PT-proteoliposomes. The G6P uptake activity decreases as the internal:external Pi ratio decreases and the Pi uptake activity decreases in the presence of external G6P. Moreover, G6P or Pi uptake activity is not detectable in Pi-loaded proteoliposomes containing the p.R28H G6PT null mutant. The G6PT-proteoliposome-mediated G6P or Pi uptake is inhibited by cholorgenic acid and vanadate, both specific G6PT inhibitors. Glucose-6-phosphatase-{alpha} (G6Pase-{alpha}), which facilitates microsomal G6P uptake by G6PT, fails to stimulate G6P uptake in Pi-loaded G6PT-proteoliposomes, suggesting that the G6Pase-{alpha}-mediated stimulation is caused by decreasing G6P and increasing Pi concentrations in microsomes. Taken together, our results suggest that G6PT has a dual role as a G6P and a Pi transporter and that GSD-Ib and GSD-Ic are deficient in the same G6PT gene.—Chen, S.-Y., Pan, C.-.J, Nandigama, K., Mansfield, B., Ambudkar, S., Chou, J. The glucose-6-phosphate transporter is a phosphate-linked antiporter deficient in glycogen storage disease type Ib and Ic.


Key Words: proteoliposomes • glucose-6-phosphatase-alpha • endoplasmic reticulum • chorogenic acid • vanadate


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
GLYCOGEN STORAGE DISEASE type I (GSD-I) is a group of autosomal recessive disorders caused by a deficiency in the endoplasmic reticulum (ER) -associated glucose-6-phosphatase-{alpha} (G6Pase-{alpha}) complex that catalyzes the hydrolysis of glucose-6-phosphate (G6P) to glucose and phosphate in the terminal step of gluconeogenesis and glycogenolysis (1) . Kinetic analyses of G6Pase-{alpha} catalysis using intact and disrupted hepatic microsomal preparations led Arion and co-workers to propose that G6Pase-{alpha} is a multicomponent, membrane-associated, enzyme complex referred to as the "G6Pase system" (2 3 4) . They proposed that the system consisted of four separate proteins: the G6Pase-{alpha} catalytic unit (also known as G6PC), the glucose-6-phosphate transporter (G6PT, also known as SLC37A4), an inorganic phosphate (Pi) transporter, and a glucose transporter. This proposal is consistent with biochemical observations, showing that one group of GSD-I patients appeared to have no G6Pase-{alpha} activity in frozen liver biopsy samples, while the other group appeared to have retained G6Pase-{alpha} activity (5 , 6) . Moreover, the active site of G6Pase-{alpha} is not accessible from the cytoplasm (7) and requires a G6PT to translocate G6P from cytoplasm into the lumen of the ER. This proposal is also consistent with the observed clinical heterogeneities, showing that one group of GSD-I patients manifest only a phenotype of disturbed glucose homeostasis while the other group exhibited a disturbed glucose homeostasis and neutrophil dysfunctions (8 , 9) . Accordingly, type I GSD was divided into four subtypes, corresponding to defects in the G6Pase-{alpha} catalytic unit, GSD-Ia (MIM232200); defects in the G6PT, GSD-Ib (MIM232220); defects in a putative Pi transporter, GSD-Ic; and defects in a putative glucose transporter, GSD-Id (reviewed in ref. 1 ). Part of this hypothetical system has been confirmed experimentally. Both the G6PC (10 , 11) and G6PT (12 , 13) genes have been cloned, and molecular genetic studies have confirmed that mutations inactivating G6Pase-{alpha} cause GSD-Ia (11 , 14 15 16) and mutations inactivating G6PT cause GSD-Ib (17 18 19) . In contrast, GSD-Ic has not been characterized at the molecular level, and the existence of the GSD-Id disease remains unproven. In sequencing the G6PT gene in clinical cases reported to represent GSD-Ic, deleterious G6PT mutations found in GSD-Ib patients were identified (20 21 22 23) . This finding raised the possibility that G6PT is implicated in both GSD-Ib and GSD-Ic, which, if correct, would imply that G6PT is a G6P and a Pi transporter.

By sequence homology, G6PT belongs to the organophosphate:phosphate antiporter family of the major facilitator superfamily (24) that also includes the glycerol-3-phosphate transporter (25) and the bacterial hexose-6-phosphate transporter, UhpT (26) . Indeed, the G6PT cDNA was originally isolated based on a comparison of the sequence of a Lactobacillus lactis hexose-6-phosphate transporter with liver expressed sequence tags (12) . Although G6PT shares only 20% amino acid sequence identity to UhpT, their hydropathy profiles are nearly superimposable (12) . Using reconstituted proteoliposomes, UhpT has been shown to be a Pi-linked antiporter capable of both homologous (Pi:Pi) and heterologous (G6P:Pi) exchange (27) . The direction of the exchange depends on the phosphate gradient. In this study, we have used a similar approach (27 28 29 30) to show that G6PT is a eukaryotic antiporter that can transport G6P into the lumen of the ER and Pi out. Our results demonstrate that G6PT has a dual role as both the G6P and the Pi transporter, providing the evidence why GSD-Ib and GSD-Ic patients harbor deleterious mutations in the same G6PT gene.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Gene expression in COS-1 cells and microsomal G6P uptake assays
Recombinant adenovirus carrying wild-type G6PT (Ad-G6PT), p.R28H G6PT mutant (Ad-G6PT-R28H), wild-type G6Pase-{alpha} (Ad-G6Pase-{alpha}), or p.H176A G6Pase-{alpha} mutant (Ad-G6Pase-{alpha}-H176A) have been described (19 , 31) . The recombinant virus was plaque purified and amplified to produce viral stocks with titers of ~1 to 3 x 1010 plaque forming units (PFU) per milliliter.

COS-1 cells were grown at 37°C in HEPES-buffered Dulbecco’s modified minimal essential medium supplemented with 4% fetal bovine serum. Cells in 150 cm2 flasks were infected with Ad-G6PT, Ad-G6PT-R28H, Ad-G6Pase-{alpha}, or Ad-G6Pase-{alpha}-H176A, or coinfected with Ad-G6PT/Ad-G6Pase-{alpha} or Ad-G6PT/Ad-G6Pase-{alpha}-H176A. The multiplicity of infection (MOI) for Ad-G6PT or Ad-G6PT-R28H was 50 PFU/cell, and the MOI for Ad-G6Pase-{alpha} or Ad-G6Pase-{alpha}-H176A was 25 PFU/cell. Mock-infected COS-1 cells were used as controls. After incubation at 37°C for 24 h, the infected cultures were used to isolate microsomes for G6P uptake, proteoliposome reconstitution, and Western blot analysis.

Microsomal G6P uptake measurements were performed essentially as described previously (17) . Briefly, microsomes (40 µg) were incubated in a reaction mixture (100 µl) containing 50 mM sodium cacodylate buffer, pH 6.5; 250 mM sucrose; and 0.2 mM [U-14C] G6P (50 µCi/µmol, American Radiolabeled Chemicals, St. Louis, MO, USA). The reaction was stopped at the appropriate time by filtering immediately through a nitrocellulose membrane (0.45 µm, Millipore Co., Billerica, MA, USA), washed with an ice-cold solution containing 50 mM Tris-HCl, pH 7.4, and 250 mM sucrose. The dried filters were counted in a liquid scintillation counter. In microsomes expressing both G6Pase-{alpha} and G6PT, the [U-14C]G6P taken up by the microsomes is hydrolyzed by G6Pase-{alpha} to [U-14C]glucose and Pi, and the radioactive molecules accumulated inside the microsomes are primarily [U-14C]glucose (32) .

Solubilization and reconstitution of membrane proteins
Microsomal membrane protein solubilization and proteoliposome reconstitution were performed as described previously (27 28 29 30) , with modifications. Briefly, membrane proteins were solubilized on ice by mixing 2 mg of microsomes in 1 ml of a solution containing 20 mM Tris-HCl, pH 7.5; 20% glycerol; 1.25% (w/v) n-octyl-β-D-glucopyranoside (octylglucoside; Calbiochem, San Diego, CA, USA); 2 mM DTT; protease inhibitors (1% aprotinin, 1 mM AEBSF, 2 µg/ml pepstatin A, and 2 µg/ml leupeptin, all from Roche Diagnostics, Indianapolis, IN, USA); and 0.4% (w/v) lipid mixture in 2 mM β-mercaptoethanol containing the Escherichia coli polar lipid extract, L-{alpha}-phosphotidylcholine, L-{alpha}-phosphotidylserine, and cholesterol (60:17.5:10:12.5 w/w), all from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). After 20 min gentle stirring, the insoluble materials were pelleted by centrifugation at 170,200 g for 60 min at 4°C. Aliquots of the clear supernatant were stored at –70°C.

The sonicated lipid mixture used for proteoliposome reconstitution was prepared by transferring 40–60 mg of lipid mixture to a Pyrex tube and sonicating in a bath-type sonicator (Laboratory Supplies Inc., Hicksville, NY, USA) at 18–23°C until the solution appeared clear, as described previously (29) .

To reconstitute proteoliposomes, the detergent-solubilized microsomal membrane extracts (500 to 700 µg) were mixed with the sonicated lipid mixture at a ratio of 1:10 protein:lipid (w/w) and the reaction brought to a final volume of 4 ml by the addition of octylglucoside dilution buffer (100 mM KPO4, pH 7.5; 1.25% octylglucoside; 2 mM DTT; and 1% aprotinin). After mixing, the suspension was placed on ice for 20 min. Proteoliposomes were then formed by placing the reaction inside a dialysis cassette (Pierce, Rockford, IL, USA) and dialyzing extensively overnight against a phosphate buffer (10, 20, or 50 mM KH2PO4, pH 7.0; 1 mM DTT; and protease inhibitors) or a MOPS/K buffer (20 mM MOPS, pH 7.5, adjusted with KOH; 75 mM K2SO4; 2.5 mM MgSO4; 1 mM DTT; and protease inhibitors) at 4°C. The resulting Pi-loaded or MOPS-loaded proteoliposomes were pelleted by centrifugation at 170,200 g for 60 min at 4°C, resuspended in 200 µl MOPS/K buffer, and used in transport assays. Proteoliposomes prepared from detergent-solubilized microsomal membrane extracts of mock-infected cells and liposomes lacking microsomal proteins were prepared in parallel and used as negative controls. The protein content in microsomes, detergent extract, and proteoliposomes was quantified by the Amido Black B protein estimation method as described previously (33) .

Phosphohydrolase assays
G6Pase-{alpha} phosphohydrolase activity was determined essentially as described previously (10 , 11) . Reaction mixtures (100 µl) contained 50 mM cacodylate buffer, pH 6.5; 10 mM G6P; 2 mM EDTA; and appropriate amounts of membrane proteins and were incubated at 30°C for 10 min. Sample absorbance was determined at 820 nm and was related to the amount of Pi released using a standard curve constructed from serial dilutions of a stock of Pi solution.

Western blot analysis
For Western blot analysis, proteins were resolved by electrophoresis through a 10% polyacrylamide-SDS gel and transblotted onto polyvinylidene fluoride membranes (Millipore). The membranes were incubated overnight with a rabbit anti-G6PT antibody (19) or a mouse monoclonal antibody raised against amino acids 81 to 119 of human G6Pase-{alpha}. The membranes were then incubated with the appropriate horseradish peroxidase-conjugated second antibody, and the immunocomplex was visualized using the ImmobilonTM Western chemiluminescent HRP substrate (Millipore).

Transport assays
Transport assays were performed at room temperature for various lengths of time using reaction mixtures containing 20 mM MOPS/K buffer, 25 µg/ml of Pi- or MOPS-loaded proteoliposomes (or Pi- or MOPS-loaded liposomes), and 0.1 mM [U-14C]G6P (50 µCi/µmol) or 0.5 mM 32Pi. The 32Pi was prepared by boiling carrier-free 32Pi (MP Biochemical, Inc., Irvine, CA, USA) in 1 ml of 1 N HCl for 3 h and diluting with an equal volume of 2 M K2HPO4 as described previously (34) . At each of the assay time points (1, 3, 6, 9, and 20 min), 100 µl aliquots were withdrawn, filtered immediately through presoaked 0.22 µm nitrocellulose filters (Millipore), and washed with 20 mM MOPS/K buffer. The dried filters were then counted in a liquid scintillation counter. Inhibition of G6P or Pi transport by chlorogenic acid (CHA) (35) or vanadate (36 , 37) was examined in reaction mixtures in 20 mM MOPS/K buffer containing 25 µg/ml of 50 mM Pi,-loaded G6PT-proteoliposomes, 0.1 mM [U-14C]G6P or 0.5 mM 32Pi, and the respective inhibitor. After incubation at room temperature for 9 min, the reaction mixture was filtered and counted as described above.

Statistical analysis
The unpaired t test was performed using GraphPad Prism, version 4 (GraphPad Software, San Diego, CA, USA). Values were considered statistically significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Solubilization and reconstitution of membrane proteins
To examine the G6P transport activity of G6PT, proteoliposomes were reconstituted from detergent-solubilized microsomal membrane extracts isolated from COS-1 cells infected with the Ad-G6PT construct that directs high levels of G6PT expression (19) . Control proteoliposomes were reconstituted from detergent-solubilized microsomal membrane extracts isolated from COS-1 cells infected with either the Ad-G6Pase-{alpha} construct or the Ad-G6PT-R28H construct that expresses a G6PT mutant lacking the G6P transport function (19) . Coinfection controls were reconstituted from COS-1 cells coinfected with Ad-G6PT and Ad-G6Pase-{alpha}. Since COS-1 cells express the G6PT gene, albeit at low levels, proteoliposomes, reconstituted from detergent-solubilized microsomal membrane extracts isolated from mock-infected COS-1 cells, were added as additional controls.

During proteoliposome preparation, solubilization and reconstitution were monitored by Western blot analysis of G6PT and/or G6Pase-{alpha}. However, both G6PT (38) and G6Pase-{alpha} (39) are highly hydrophobic proteins anchored to the ER by multiple transmembrane helices. To ensure that these membrane proteins remained folded correctly and functional, the solubilization and reconstitution of the G6Pase-{alpha} enzyme were also monitored. The overall yields of proteins in octylglucoside extracts and reconstituted proteoliposomes were 32 and 12%, respectively, and total G6Pase-{alpha} activity recovered in octylglucoside extracts and proteoliposomes was 52–53 and 38–42%, respectively (Table 1 ). The specific activity of G6Pase-{alpha} in the octylglucoside extracts increased 1.6-fold over the activity isolated from the Ad-G6Pase-{alpha}-infected or Ad-G6PT/Ad-G6Pase-{alpha}-coinfected COS-1 microsomes, while the specific activity in reconstituted proteoliposomes increased 3.2- to 3.5-fold (Table 1) , indicating enrichment of the G6Pase-{alpha} protein. These increases in G6Pase-{alpha} specific activity correlated with the increase in the G6Pase-{alpha} protein visualized on Western blots (Fig. 1 ), indicating that biologically active membrane proteins were solubilized and reconstituted. Western blot analysis showed similar enrichments in G6PT proteins in octylglucoside extracts and proteoliposomes (Fig. 1) .


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  		Table 1. Recovery of G6Pase-{alpha} activity in proteoliposomes reconstituted with detergent-solubilized microsomal membrane extracts


Figure 1
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  		Figure 1. Western blot analysis of G6PT and G6Pase-{alpha}. Microsomes were isolated from COS-1 cells infected with Ad-G6PT, Ad-G6Pase-{alpha}, or Ad-G6PT-R28H, or coinfected with Ad-G6PT and Ad-G6Pase-{alpha}. Microsomal membrane proteins were solubilized with octylglucoside in the presence of lipid and glycerol, as described in Materials and Methods. The proteoliposomes were prepared by mixing octylglucoside-solubilized microsomal proteins with a sonicated lipid mixture and then dialyzing overnight against a 50 mM phosphate (proteoliposomes/Pi) or MOPS/K (proteoliposomes/MOPS) buffer, as described in Materials and Methods. Proteins were resolved by SDS-gel electrophoresis, transblotted onto polyvinylidene fluoride membranes, and analyzed using a rabbit anti-G6PT antibody (19) or a mouse monoclonal antibody raised against amino acids 81 to 119 of human G6Pase-{alpha}. Each lane contains 10 µg protein.

We have previously shown that the optimal MOI of Ad-G6PT and Ad-G6Pase-{alpha} for microsomal G6P uptake activity is 50 and 25, respectively (19) . At a MOI of 25, G6Pase-{alpha} specific activity was 335.1 ± 8.8 nmol/min/mg in Ad-G6Pase-{alpha}-infected microsomes but 253.7 ± 9.2 nmol/min/mg in Ad-G6PT/Ad-G6Pase-{alpha}-coinfected microsomes (Table 1) , suggesting that the expression efficiency of G6Pase-{alpha} was inhibited by the coinfected Ad-G6PT. At a MOI of 25, Western blot analysis showed that the amounts of G6Pase-{alpha} proteins in Ad-G6Pase-{alpha}-infected microsomes were higher than those observed in Ad-G6PT/Ad-G6Pase-{alpha}-coinfected microsomes (Fig. 1) , confirming such inhibition. Similarly, expression of the G6PT protein was inhibited by the coinfected Ad-G6Pase-{alpha} (Fig. 1) . However, the efficiency of solubilization and reconstitution of G6PT under all experimental conditions remained unchanged.

G6PT mediates heterologous exchanges of G6P and Pi
To investigate whether G6PT can mediate heterologous exchanges of G6P and Pi, G6P uptake was examined in proteoliposomes reconstituted from Ad-G6PT-infected microsomes loaded with MOPS (0 mM Pi) or 10, 20, or 50 mM Pi (Fig. 2 A). G6P was efficiently taken up in G6PT-proteoliposomes loaded with 50 mM Pi, but only very low levels of G6P transport were seen in the MOPS-loaded (0 mM Pi) G6PT-proteoliposomes. Consistent with the findings for UhpT (29) , the rate of G6P uptake correlated with the concentration of Pi loaded in the G6PT-proteoliposomes (Fig. 2A ). Moreover, a very low level of G6P uptake activity was detected in 50 mM Pi-loaded proteoliposomes reconstituted from detergent-solubilized microsomal membrane extracts isolated from COS-1 cells infected by the null mutant Ad-G6PT-R28H (Fig. 2B ). The low levels of G6P accumulation in G6PT-R28H-proteoliposomes represent background activity because similar activities were observed using proteoliposomes reconstituted from detergent-solubilized microsomal membrane extracts isolated from mock-infected COS-1 cells (data not shown).


Figure 2
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  		Figure 2. Heterologous exchange of G6P and Pi in proteoliposomes. Proteoliposomes were reconstituted from detergent-solubilized microsomal membrane extracts isolated from Ad-G6PT or Ad-G6PT-R28H infected COS-1 cells as described under Materials and Methods. Results shown are from three independent experiments, each point determined in triplicate. A) Effects of Piloading on G6P uptake activity of proteoliposomes containing G6PT. The concentrations of Pi loaded are 50 ({circ}), 20 (•), 10 ({triangledown}), and 0 mM (MOPS, {blacktriangledown}). B) Inhibition of G6P uptake in 50 mM Pi-loaded G6PT-proteoliposomes ({circ}) by 50 ({triangledown}) and 100 mM external Pi ({blacktriangledown}). G6P uptake in the G6PT-R28H-proteoliposomes loaded with 50 mM Pi (•) is also shown. Data are presented as means ± SE.

The antiporter activity of the G6PT was further examined in 50 mM Pi-loaded G6PT-proteoliposomes in the presence of various external concentrations of Pi. Results showed that the addition of external Pi markedly inhibited G6P uptake (Fig. 2B ). The 20 min uptake rate was 2.72 ± 0.20 in G6PT-proteoliposomes without external Pi, but the rate decreased to 0.71 ± 0.02 and 0.19 ± 0.3 in the presence of 50 and 100 mM external Pi, respectively (Fig. 2B ). In fact, G6P uptake activity in 50 mM Pi-loaded G6PT-proteoliposomes in the presence of 100 mM external Pi was lower than the activity observed in the MOPS-loaded G6PT-proteoliposomes (data not shown) or in 50 mM Pi-loaded proteoliposomes containing the G6PT-R28H null mutant (Fig. 2B ).

G6PT mediates homologous Pi:Pi exchanges
UhpT is a P-linked antiporter that can also mediate homologous Pi:Pi exchanges (27) . We therefore examined Pi uptake in G6PT-proteoliposomes loaded with MOPS (0 mM Pi) or 20 or 50 mM Pi. The Pi was efficiently taken up in G6PT-proteoliposomes loaded with 50 mM Pi, but uptake activity was markedly reduced in G6PT-proteoliposomes loaded with 20 mM Pi, which become background levels in MOPS-loaded (0 mM Pi) G6PT-proteoliposomes (Fig. 3 A). As was demonstrated for G6P uptake, only very low levels of Pi uptake activity were detected in G6PT-R28H-proteoliposomes loaded with 50 mM Pi (Fig. 3A ). Again, similar levels of Pi uptake were observed with proteoliposomes reconstituted from detergent-solubilized microsomal membrane extracts isolated from Ad-G6PT-R28H- or mock-infected COS-1 cells (data not shown).


Figure 3
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  		Figure 3. Homologous exchange of Pi:Pi in proteoliposomes. Proteoliposomes were reconstituted from detergent-solubilized microsomal membrane extracts isolated from Ad-G6PT- or Ad-G6PT-R28H-infected COS-1 cells, as described in Materials and Methods. Results shown are from three independent experiments, each point determined in triplicate. A) Effects of Piloading on Pi uptake activity of proteoliposomes containing G6PT. The concentrations of Pi loaded are 50 ({circ}), 20 (•), and 0 mM (MOPS, {blacktriangledown}). G6P uptake in the G6PT-R28H-proteoliposomes loaded with 50 mM Pi ({triangledown}) is also shown. B) Inhibition of Pi uptake in 50 mM Pi-loaded G6PT-proteoliposomes by external G6P. Data are presented as means ± SE. **P < 0.001.

The antiporter activity of the G6PT was then examined in 50 mM Pi-loaded G6PT-proteoliposomes by external G6P. As expected, the addition of external G6P markedly inhibited Pi uptake (Fig. 3B ). The Pi uptake activity was inhibited by 68, 79, 86, and 89% by 1, 5, 10, and 20 mM of external G6P, respectively (Fig. 3B ).

Inhibition by CHA and vanadate
To further elucidate antiporter activity of the G6PT protein, we examined the effects of CHA, a specific inhibitor of G6PT (35) , and vanadate, a close structural and chemical mimic of the phosphate ion (36 , 37) . Vanadate is a potent inhibitor of enzymes that catalyze phosphoryl transfer reactions (36 , 37) and has been shown to inhibit G6P transport in hepatic microsomes (40) . Results in Fig. 4 A show that G6P uptake in Pi-loaded G6PT-proteoliposomes was inhibited 40% by 1 mM CHA and 85% by 2 mM CHA. Likewise, Pi uptake in Pi-loaded G6PT-proteoliposomes was inhibited 45% by 1 mM CHA and 85% by 2 mM CHA (Fig. 4A ).


Figure 4
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  		Figure 4. Inhibition of G6P and Pi transport in G6PT-expressing proteoliposomes by CHA and vanadate. Proteoliposomes were reconstituted from detergent-solubilized microsomal membrane extracts expressing G6PT and were loaded with 50 mM Pi, as described in Materials and Methods. Results shown are from three independent experiments, each point determined in triplicate. A) Effects of CHA on G6P and Pi uptake. B) Effects of vanadate on G6P and Pi uptake. Data are presented as means ± SE. *P < 0.05; **P < 0.005.

Similarly, G6P uptake in Pi-loaded G6PT-proteoliposomes was inhibited 34% by 1 mM vanadate and 79% by 2 mM vanadate, and Pi uptake was inhibited 53% by 1 mM vanadate and 91% by 2 mM vanadate (Fig. 4B ).

Effects of G6Pase-{alpha} on G6P transport
We have previously shown that G6Pase-{alpha} facilitates microsomal G6P transport by the G6PT (17 , 40) . In vitro expression studies have shown that G6PT-expressing microsomes exhibit only low levels of G6P transport activity but microsomes expressing both G6PT and G6Pase-{alpha} have markedly increased transport activity (Fig. 5 A). However, microsomes expressing the G6Pase-{alpha}-H176A mutant or expressing both G6PT and G6Pase-{alpha}-H176A exhibit only background G6P transport activity (Fig. 5A ). In G6Pase-{alpha}, His-176 is the phosphate acceptor, and the H176A mutant is devoid of G6P hydrolase activity (39) . Thus, the enhancement of G6PT-mediated microsomal G6P uptake may result from the hydrolytic activity of G6Pase-{alpha}, which converts endoluminal G6P to glucose and Pi, generating a driving Pi gradient (27 , 30) .


Figure 5
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  		Figure 5. Effects of G6Pase-{alpha} on G6P transport activity of G6PT. Microsomal membranes were isolated from COS-1 cells infected with Ad-G6PT or Ad-G6Pase-{alpha}-H176A, or coinfected with Ad-G6PT/Ad-G6Pase-{alpha} or Ad-G6PT/Ad-G6Pase-{alpha}-H176A, as described in Materials and Methods. Mock-infected microsomes were used as controls. Proteoliposomes were reconstituted from the respective detergent-solubilized microsomal membrane extracts and loaded with 50 mM Pi, as described in Materials and Methods. Results represent three independent experiments, with each data point determined

in triplicate. Data are presented as means ± SE. A) Microsomal G6P uptake activity. *P < 0.05, indicated comparisons;

**P < 0.005, G6PT/G6Pase-{alpha} vs. all other conditions. B) Proteoliposomal G6P uptake activity. *P < 0.05; **P < 0.005, G6PT vs. all other conditions and other indicated comparisons.

In the case of the Pi-loaded G6PT-proteoliposomes, it was anticipated that the increased Pi concentration arising from G6P hydrolysis would be small compared to the driving Pi gradient created during the proteoliposome formation. Moreover, high Pi concentrations inhibit G6Pase-{alpha} catalytic activity (41) ; therefore, the G6Pase-{alpha} coexpression would not improve G6P transport in these experiments. This was confirmed experimentally, as shown in Fig. 5B . While G6P uptake activity in Pi-loaded G6Pase-{alpha}-H176A-proteoliposomes was low at background levels, G6P uptake activity in Pi-loaded proteoliposomes containing G6PT and the G6Pase-{alpha}-H176A mutant was slightly higher than the activity in Pi-loaded proteoliposomes containing G6PT and G6Pase-{alpha} (Fig. 5B ). Unlike microsomes, which are right-side-out, the orientation of the protein reconstituted in the proteoliposomes is random (42) , and the active site of G6Pase-{alpha} is located at both sides of the proteoliposomes. The apparent inhibition of proteoliposome-mediated G6P uptake by G6Pase-{alpha} may result from a reduced expression efficiency of G6PT by the coinfected Ad-G6Pase-{alpha} or Ad-G6Pase-{alpha}-H176A (Fig. 1) , as well as by decreasing the external G6P concentrations by G6Pase-{alpha}.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
When originally characterized, GSD-I was proposed to consist of 4 subtypes: GSD-Ia, which is deficient in the G6Pase-{alpha} catalytic unit; GSD-Ib, which is deficient in the G6PT; GSD-Ic, which is deficient in a Pi transporter; and GSD-Id, which is deficient in a glucose transporter (1) . These proposals were supported by kinetic studies of G6Pase-{alpha} catalysis (2 3 4) as well as by biochemical and clinical heterogeneities observed in GSD-I patients (5 6 7 8 9) . More recently, molecular genetic studies have clearly confirmed the genetic basis for GSD-Ia (11) and GSD-Ib (12 , 13) , but the genetic basis for GSD-Ic and GSD-Id remains unexplored. In this study, we explored the possibility that deficiencies in G6PT also form the basis of GSD-Ic. The rationale was 2-fold. First, G6PT shares sequence identity with UhpT, a Pi-linked Pi:Pi and G6P:Pi antiporter (26 , 27) . Indeed, the G6PT cDNA was first cloned based on this potential similarity (12) . Second, most, if not all, patients previously identified as GSD-Ic harbor mutations in the G6PT gene (20 21 22 23) , including c.1042_1043delCT, the prevalent mutation found in GSD-Ib patients (43) . Moreover, the 10 missense G6PT mutations identified (20 21 22 23) , including the four mutations p.Q133P, p.R300C, p.A367T, and p.G376S, found only in GSD-Ic patients to date, were all shown to abolish or markedly reduce microsomal G6P transport activity (19) . Our working hypothesis was that G6PT has a dual transport role for both G6P and Pi. Using reconstituted G6PT-proteoliposomes, we show that G6PT is an antiporter that mediates homologous Pi:Pi and heterologous G6P:Pi exchanges, providing one explanation why clinically reported GSD-Ic patients harbor deleterious mutations in the G6PT gene. Our study reconciles the reclassification of GSD-Ic as GSD-Ib (20 21 22 23) .

Using reconstituted G6PT-proteoliposomes, we show that G6P or Pi uptake occurs in Pi-loaded proteoliposomes and that the rates of G6P or Pi uptake correlates positively with the concentration of the Pi loaded into the proteoliposomes. Supporting the role of G6PT specifically as the transporter in this system, we also show that the Pi-loaded proteoliposomes containing the G6PT-R28H null mutant lack G6P or Pi transport activity. Moreover, the reconstituted proteoliposome system retains the characteristics of the G6PT as CHA, a specific inhibitor of the G6PT (35) , suppresses G6P and Pi transport in Pi-loaded G6PT-proteoliposomes (see Fig. 4 ). Similarly, vanadate, a close structural and chemical mimic of phosphate (37) , also inhibits G6P and Pi transport in Pi-loaded G6PT-proteoliposomes. Taken together, our results, for the first time, demonstrate that G6PT, like UhpT, is a Pi-linked G6P and Pi transporter.

The question remains why G6Pase-{alpha} stimulates G6P transport by the G6PT in microsomes. The answer may lie in the products of G6P hydrolysis by G6Pase-{alpha}, namely glucose and phosphate, as well as the nature of microsomes, which are very small structures with a high surface-to-volume ratio (44) . As hydrolysis occurs, Pi increases in the lumen of the ER and generates a driving Pi gradient that leads to an increase in G6P uptake mediated by the heterologous antiporter activity of G6PT. The small volume of the ER would suggest that this control could be very finely tuned to balance the needs of the ER by the G6Pase-{alpha} activity. This was supported by the finding that G6PT-mediated microsomal G6P transport was not stimulated by the G6Pase-{alpha}-H176A null mutant.

While some GSD-Ic patients have been shown to harbor mutations in the G6PT gene, this may not be the sole source of Pi transport out of the ER. At least one patient classified as GSD-Ic based on kinetic analyses of the hepatic G6Pase-{alpha} system (45) has been shown to lack mutations in either the G6PC (15) or the G6PT (46) gene. This finding could be taken to suggest the existence of an additional ER-associated Pi transporter. However, this patient was originally classified GSD-I more by default than by positive scoring. The patient presented with diabetes, hepatomegaly, and increased hepatic glycogen deposition in the absence of disorders associated with other forms of GSD—normal hepatic branching enzyme, debranching enzyme, lysosomal {alpha}-glucosidase, and fructose-diphosphatase activities (45) . Presently, whether this patient is defective in a separate ER-associated Pi transporter remains to be elucidated.

In summary, we have shown that G6PT is an antiporter that mediates homologous Pi:Pi as well as heterologous G6P:Pi exchanges and functions as a G6P and a Pi transporter of the ER. Our study verifies the existence of an ER-associated Pi transporter predicted by kinetic studies (2 3 4) and confirms that GSD-Ib and GSD-Ic are deficient in the same G6PT gene. The reconstituted proteoliposome system can now be used to characterize G6PT mutations identified in GSD-Ib/GSD-Ic patients and structure-function studies of this transporter.


   ACKNOWLEDGMENTS
 
This research was supported in part by the Intramural Research Programs of the National Institute of Child Health and Human Development and the National Cancer Institute, U.S. National Institutes of Health.

Received for publication December 19, 2007. Accepted for publication February 7, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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J. C. Hutton and R. M. O'Brien
Glucose-6-phosphatase Catalytic Subunit Gene Family
J. Biol. Chem., October 23, 2009; 284(43): 29241 - 29245.
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