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Originally published In Press as doi:10.1074/jbc.M502483200 on May 11, 2005

J. Biol. Chem., Vol. 280, Issue 26, 24301-24307, July 1, 2005
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Physical and Kinetic Interactions between Glutamyl-tRNA Reductase and Glutamate-1-semialdehyde Aminotransferase of Chlamydomonas reinhardtii*

Luiza A. Nogaj and Samuel I. Beale{ddagger}

From the Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912

Received for publication, March 7, 2005 , and in revised form, April 28, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In plants, algae, and most bacteria, the heme and chlorophyll precursor 5-aminolevulinic acid (ALA) is formed from glutamate in a three-step process. First, glutamate is ligated to its cognate tRNA by glutamyl-tRNA synthetase. Activated glutamate is then converted to a glutamate 1-semialdehyde (GSA) by glutamyl-tRNA reductase (GTR) in an NADPH-dependent reaction. Subsequently, GSA is rearranged to ALA by glutamate-1-semialdehyde aminotransferase (GSAT). The intermediate GSA is highly unstable under physiological conditions. We have used purified recombinant GTR and GSAT from the unicellular alga Chlamydomonas reinhardtii to show that GTR and GSAT form a physical and functional complex that allows channeling of GSA between the enzymes. Co-immunoprecipitation and sucrose gradient ultracentrifugation results indicate that recombinant GTR and GSAT enzymes specifically interact. In vivo cross-linking results support the in vitro results and demonstrate that GTR and GSAT are components of a high molecular mass complex in C. reinhardtii cells. In a coupled enzyme assay containing GTR and wild-type GSAT, addition of inactive mutant GSAT inhibited ALA formation from glutamyl-tRNA. Mutant GSAT did not inhibit ALA formation from GSA by wild-type GSAT. These results suggest that there is competition between wild-type and mutant GSAT for binding to GTR and channeling GSA from GTR to GSAT. Further evidence supporting kinetic interaction of GTR and GSAT is the observation that both wild-type and mutant GSAT stimulate glutamyl-tRNA-dependent NADPH oxidation by GTR.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
5-Aminolevulinic acid (ALA)1 is the earliest universal precursor in the tetrapyrrole biosynthesis pathway (1, 2). In plants, algae, and most bacteria, ALA is generated from glutamate and its cognate tRNA in a three-step process shown in Fig. 1 (3–5). In the first step, glutamyl-tRNA synthetase activates glutamate by ligating it to tRNAGlu (UUC) (6, 7). This step of ALA production is identical to glutamate activation for protein biosynthesis (8). In the next step, the activated glutamate is converted to glutamate 1-semialdehyde (GSA) by glutamyl-tRNA reductase (GTR) in a NADPH-dependent reaction (9). This process probably involves transfer of the glutamate moiety to the active site Cys of GTR before reduction of the carboxyl group. In the final step, GSA is rearranged to ALA by glutamate-1-semialdehyde aminotransferase (GSAT) (10).

GSA, an {alpha}-amino aldehyde, is unstable at physiological pH (11). This instability could lead to degradation of the intermediate as well as the generation of toxic products within the cells. One way of minimizing the exposure of an unstable biochemical intermediate to the intracellular medium, and also to minimize the steady-state concentration of the intermediate, is to physically channel the intermediate from one enzyme to the next through specific protein-protein interactions (12). Computer-aided modeling, based on x-ray crystallographic structures of GTR from the hyperthermophilic archaeon Methanopyrus kandleri and GSAT from the cyanobacterium Synechococcus sp. 6301, has been used to predict that the two enzymes could interact to facilitate channeling of GSA (13).

In this study, we investigated interactions between GTR and GSAT using purified recombinant enzymes from Chlamydomonas reinhardtii and specific polyclonal antibodies against each protein. Co-immunoprecipitation and sucrose gradient centrifugation results provided in vitro evidence that GTR and GSAT form a complex. In vivo cross-linking results indicate the existence of a high molecular mass complex in C. reinhardtii cells that contains both GTR and GSAT. Kinetic results showing that inactive mutant GSAT inhibits ALA formation from glutamyl-tRNA in assays containing GTR and wild-type GSAT support the conclusion that GSA is channeled between GTR and GSAT. Additional evidence for specific interaction between the two enzymes is that GSAT specifically stimulates glutamyl-tRNA-dependent NADPH oxidation catalyzed by GTR.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
C. reinhardtii Enzymes and Antibodies to the Enzymes—A full-length cDNA clone encoding GSAT was previously isolated and cloned into pBSIISK+ plasmid (14). The GSAT open reading frame without the predicted 30-residue transit peptide (14) was recloned into expression plasmid pQE30 (Qiagen, Valencia, CA) using the BamHI and SacI restriction sites and transformed into an Escherichia coli expression host SG13009 (pREP4) (Qiagen). Expression of recombinant GSAT containing N-terminal His6 tag was induced with 1 mM isopropyl {beta}-D-thiogalactopyranoside for 2 h at 37 °C. The product was purified under native conditions using Ni-NTA-agarose (Qiagen). GSAT protein was eluted from the Ni-NTA using 50 mM monobasic sodium phosphate, 300 mM sodium chloride, 250 mM imidazole, pH 8.0. The purified GSAT was used for generation of polyclonal anti-GSAT antibodies (Animal Pharm Services, Cloverdale, CA).

A catalytically active expression construct of GTR corresponding to the open reading frame encoded by exon 2 of the GTR gene and containing a C-terminal His6 tag was previously described (15). Expression of this construct was induced with 0.2 mM isopropyl-1-thio-{beta}-D-galactopyranoside for 15 h at 26 °C. The product was purified under native conditions using Ni-NTA-agarose and used for the generation of polyclonal anti-GTR antibodies as described above for GSAT (Animal Pharm Services).



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  		FIG. 1.
Steps in the conversion of glutamate to ALA.

 
Site-directed Mutagenesis of GTR—The conserved active site cysteine residue occurs at position 48 in C. reinhardtii GTR (13, 15). This residue was replaced with an alanine residue by PCR, using 5'-GGTGCTGTCGACGGCCAATCGCATGGAGC-3' and its complement as forward and reverse primers and the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The PCR products were transformed into E. coli expression strain SG13009 pREP4. The K273A GSAT was sequenced, overexpressed, and purified using Ni-NTA as described. K273A GSAT was examined for size by SDS-PAGE and for presence of a His tag with anti-His antibodies (Sigma).

Site-directed Mutagenesis of GSAT—The conserved active site lysine residue (16, 17) occurs at position 273 in C. reinhardtii GSAT. This residue was replaced with an alanine residue by polymerase chain reaction (PCR), using 5'-CCACCATGGGCGCGGTCATTGGTGGCGG-3' and its complement as forward and reverse primers and the QuikChange site-directed mutagenesis kit. The PCR products were handled as described above.

Co-immunoprecipitation—Purified GTR and GSAT proteins were incubated with anti-GTR or anti-GSAT antibodies in 0.5 ml of reaction buffer (25 mM Tris-HCl, pH 7.4, 0.5 mM EDTA, 1% (w/v) Nonidet P-40, and 10% (w/v) glycerol) for 3 h at 4 °C in silicone-coated 1.5-ml microcentrifuge tubes (USA Scientific, Ocala, FL). After addition of 20 µl of 50% (w/v) protein A-Sepharose beads (Sigma), the proteins were incubated for an additional 1 h as described (18, 19). The immunocomplexes were precipitated and washed with ice-cold reaction buffer three times, resuspended in loading buffer, and resolved on SDS-PAGE. Protein complexes were analyzed by immunoblotting.

Sucrose Gradient Centrifugation—Recombinant GTR and GSAT proteins were sedimented through a 5-ml continuous 15–35% (w/v) sucrose gradient formed in 50 mM Tricine, pH 7.9, 15 mM MgCl2. The gradients were centrifuged in a Beckman SW 50.1 rotor at 45,000 rpm for 22 h at 4 °C. After centrifugation, the gradients were fractionated by puncturing the centrifuge tubes at the bottom and collecting 0.2-ml fractions. Molecular mass standard proteins (MW-GF-200; Sigma) were co-sedimented on identical gradients during each centrifugation. Proteins in gradient fractions were measured by a dye binding assay (20).

In Vivo Cross-linking—C. reinhardtii CC124 cells grown in continuous light were used. Small aliquots (1 ml) of cell suspension were centrifuged, and the cells were resuspended in assay buffer (50 mM Tricine, pH 7.9, 1 M glycerol, 15 mM MgCl2) containing various concentrations of glutaraldehyde (Sigma) for 30 min at room temperature with constant mixing. Cross-linking reactions were stopped by repeated centrifuging and resuspending the cells in ice-cold assay buffer. After the final wash, cell pellets were resuspended and lysed in 50 mM Na2CO3, and the proteins were resolved on a 6% (w/v) SDS-PAGE gel. Cross-linked proteins were analyzed by immunoblotting using anti-GTR and anti-GSAT antibodies.

Determination of GSAT Activity—GSAT was incubated in 0.5 ml of assay buffer supplemented with 2 mM GSA, synthesized as described (11), 5 mM levulinic acid, 1 mM DL-dithiothreitol, and 20 mM pyridoxal-P, for 30 min at 30 °C. Reactions were started by adding GSA from a concentrated acidic stock solution. The reaction was stopped by adding, with mixing, 25 µl of 100% (w/v) trichloroacetic acid, and the mixture was incubated for 10 min on ice. The chilled mixture was centrifuged for 10 min at 12,000 x g, and the supernatant was decanted into 150 µl of Na3PO4. ALA was converted to ALA-pyrrole by adding 25 µl of ethyl acetoacetate and mixing, heating the solution at 100 °C for 10 min, and then cooling on ice. An equal volume of freshly made Ehrlich-Hg reagent (21) was added to the cooled solution, with mixing. ALA (as the Ehrlich salt of ALA-pyrrole) was determined by measuring the A553 with a Cary 213 spectrophotometer (Varian, Palo Alto, CA) and using {epsilon}553 = 7.2 x 104 M–1 (22).

Determination of GTR Activity by ALA Formation—GTR activity was assayed as ALA formation from glutamyl-tRNA (generated in situ) in a coupled enzyme assay carried out in 96-well microtiter plates in 0.1 ml of assay buffer supplemented with 40 µg of purified recombinant C. reinhardtii GSAT, 80 units of E. coli aminoacyl-tRNA synthetases (Sigma), 0.01 units of E. coli tRNAGlu (Sigma), 5 mM ATP, 5 mM levulinic acid, 1 mM NADPH, 1 mM glutamate, 1 mM dithiothreitol, and 20 µM pyridoxal-P. Reactions were started by adding tRNA. The assay mixtures were incubated for 30 min at 30 °C. Reactions were stopped and ALA was determined as described above for GSAT but scaled down 5-fold. Light absorption was measured on a VMax Kinetic microplate reader driven by SoftMax Pro software (Molecular Devices, Sunnyvale, CA). The light absorption was measured at 562 nm, which was as close to the 553-nm absorption maximum as the instrument could attain. From the absorption spectrum of a solution of the Ehrlich salt of ALA-pyrrole and the published {epsilon}553 = 7.2 x 104 M–1 (22), a value of {epsilon}562 = 5.8 x 104 M–1 was calculated.

Determination of GTR Activity by NADPH Oxidation—GTR activity was assayed as glutamyl-tRNA-dependent NADPH oxidation in 0.5 ml of assay buffer supplemented with 400 units of E. coli aminoacyl-tRNA synthetases, 0.04 units of E. coli tRNAGlu, 5 mM ATP, 5 mM levulinic acid, 1 mM glutamate, 1 mM dithiothreitol, and 20 µM pyridoxal-P. The reaction mixture was preincubated for 5 min at room temperature to generate glutamyl-tRNA. The reaction was initiated by addition of 10 µl of 10 mM NADPH. The NADPH concentration was determined every 30 s for 15 min by measuring the A340 using an HP5402 diode-array spectrophotometer (Hewlett-Packard, Palo Alto, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Co-immunoprecipitation of GTR and GSAT—Specific polyclonal antibodies against recombinant C. reinhardtii GTR and GSAT were used in co-immunoprecipitation experiments. Each antibody was able to precipitate both proteins when added to mixtures of GTR and GSAT (Fig. 2). Neither antibody precipitated the noncognate protein when the cognate protein was absent, and the GTR-GSAT protein mixture was not precipitated in the absence of antibody.

Co-sedimentation of GTR and GSAT—The apparent native molecular masses of GTR and GSAT were determined by sucrose gradient sedimentation (Fig. 3). Linear regression interpolation from the sedimentation data indicated an apparent native molecular mass of 101 kDa for GTR and 124 kDa for GSAT. Although these values differ somewhat from the calculated molecular masses of native dimeric GTR (105 kDa) and dimeric GSAT (92 kDa), it is clear that both proteins sediment as dimers rather than monomers or higher multimers. When GTR and GSAT were preincubated together before sucrose gradient sedimentation, both proteins sedimented to a position corresponding to a molecular mass of 173 kDa. The calculated molecular mass for a 1:1 complex of native GTR and GSAT dimers is 197 kDa. These results indicate that native GTR and GSAT form a 1:1 complex of native dimers that is stable to sedimentation.

GTR and GSAT Are Part of a Large Complex in Vivo—C. reinhardtii cells grown in continuous light were treated with varying concentrations of glutaraldehyde. Immunoblots show that as the glutaraldehyde concentration was increased, bands corresponding to GTR and GSAT subunit polypeptides disappeared (Fig. 4). At increasingly higher glutaraldehyde concentrations, there was increasing intensity of a band that reacted to both anti-GTR and anti-GSAT at a molecular mass >200 kDa. On immunoblots that were simultaneously probed with both anti-GTR and anti-GSAT antibodies, a single band was observed at the position that corresponded to the >200-kDa band observed in the blots that were probed with the individual antibodies. A weak band was observed in untreated control samples at a position just below that of the glutaraldehyde-induced >200-kDa band, but this band disappeared at higher glutaraldehyde concentrations as the >200-kDa band appeared. This band was also observed on Coomassie Blue-stained gels (data not shown) and is likely because of an abundant protein that reacts nonspecifically with the antibodies. A prominent 130-kDa band was visible in all lanes of all blots and is of unknown cause, but it is unlikely to be attributable to a GTR·GSAT complex because it was also observed in control samples that were not treated with glutaraldehyde. It should be noted that in this experiment in which whole cells were treated with glutaraldehyde, most of the proteins would be expected to be cross-linked nonspecifically and most of the cross-linked products would appear as smears of undefined molecular mass or be lost from the analysis as large insoluble aggregates. Therefore, the discrete bands observed on the immunoblots should be considered to represent only the small fraction of the total proteins that reacted specifically.



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  		FIG. 2.
Co-immunoprecipitation of GTR and GSAT. A, purified recombinant GTR and GSAT proteins were incubated with anti-GTR antibody and protein A-Sepharose, and the precipitate was analyzed by immunoblot using anti-GSAT antibody. Lane 1, the complete incubation. Lanes 2–5, negative control incubations with the omission of protein A-Sepharose, anti-GTR antibody, GSAT, and GTR, respectively. Lane 6, positive control using GSAT. B, purified recombinant GTR and GSAT proteins were incubated with anti-GSAT antibody and protein A-Sepharose, and the precipitate was analyzed by immunoblot using anti-GTR antibody. Lane 1, the complete incubation; lanes 2–5, negative control incubations with the omission of protein A-Sepharose, anti-GSAT antibody, GSAT, and GTR, respectively. Lane 7, positive control using GTR. Lane 6 contains molecular mass markers that were not referred to in this experiment.

 
Free GTR disappeared even at the lowest glutaraldehyde concentration used (0.01%, w/v), but free GSAT disappeared completely only at glutaraldehyde concentrations above 0.05% (w/v). This difference suggests that there is a greater molar abundance of GSAT over GTR in the cells and is consistent with relative abundance results obtained from quantitative immunoblots (data not shown).

The >200-kDa molecular mass band that contained GTR and GSAT was probed for the presence of two other proteins. FLP is a C. reinhardtii membrane-associated protein that interacts with GTR in vitro (23) and is homologous to the Arabidopsis thaliana protein FLU that is a negative regulator of ALA synthesis (24, 25). C. reinhardtii chloroplast-specific glutamyl-tRNA synthetase has been reported to form a complex with GTR in vitro under certain conditions (26). Immunoblots of extracts from control and glutaraldehyde-treated cells were probed with antibodies to C. reinhardtii FLP and to barley chloroplast glutamyl-tRNA synthetase (27). FLP and glutamyl-tRNA synthetase were both detected in whole cell extracts of control incubations, but neither was detected in the >200-kDa band that contained GTR and GSAT in extracts of glutaraldehyde-treated cells (data not shown).



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  		FIG. 3.
Sucrose density gradient ultracentrifugation. Recombinant GTR and GSAT proteins were incubated separately or together in an assay buffer supplemented with pyridoxal-P and dithiothreitol and subjected to sucrose density gradient sedimentation. Fractions (0.2 ml) were collected from the punctured bottoms of the centrifuge tubes, and their protein content was determined by a dye binding assay. A, elution profiles are shown for GTR (circles), GSAT (squares), and the mixture of GTR and GSAT (triangles). B, peak elution volumes are shown for GTR (circle), GSAT (square), the mixture of GTR and GSAT (triangle), and the reference proteins (X), which were carbonic anhydrase (29 kDa), bovine serum albumin (66 kDa), alcohol dehydrogenase (150 kDa), and {beta}-amylase (200 kDa). The values shown are averages for six replicates.

 
GSAT Stimulates NADPH Oxidation by GTR—Because the GTR reaction involves oxidation of NADPH, it is possible to measure GTR activity in a NADPH oxidation assay that does not depend on the conversion of the reaction product, GSA, to ALA by GSAT. NADPH was slowly oxidized in the absence of any enzyme (Fig. 5). The same low background level of NADPH oxidation was observed when wild-type or K273A GSAT was added to assay mixtures in the absence of GTR. In assay mixtures containing GTR but without GSAT, the rate of NADPH oxidation was approximately twice the background level; this rate difference over the background rate was constant over the 15-min assay time. Addition of either wild-type or K273A GSAT to assay mixtures containing GTR stimulated NADPH oxidation ~2.5-fold over the level in the presence of GTR alone. In complete assay mixtures containing inactive C48R GTR instead of wild-type GTR, only background levels of NADPH oxidation occurred. In control experiments, in assay mixtures containing GTR and GSAT but with glutamate omitted, there was very little NADPH oxidation. Assay mixtures with tRNA omitted had a slight increase in NADPH oxidation rate above the background level. However, addition of RNase A to the reaction mixture brought the NADPH oxidation rate to background level. Therefore, this increase of NADPH oxidation rate by GTR in the absence of tRNA suggests that some tRNA co-purified with GTR and that this tRNA could sustain a low rate of NADPH oxidizing activity. The ability of GSAT to stimulate GTR activity supports the hypothesis that the two enzymes interact during catalysis.



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  		FIG. 4.
In vivo cross-linking of C. reinhardtii cells. Cells were grown in continuous light. Culture aliquots (1 ml) were centrifuged, and the cells were resuspended in various concentrations of glutaraldehyde for 30 min at room temperature. The cells were washed with ice-cold assay buffer and lysed in 50 mM Na2CO3, and the proteins were separated on 6% SDS-PAGE. Cross-linked proteins were detected by immunoblot with either anti-GSAT antibody, anti-GTR antibody, or both antibodies as indicated. The glutaraldehyde concentrations were: lane 1, 0%; lane 2, 0.01%; lane 3, 0.03%; lane 4, 0.05%; lane 5, 0.10%; and lane 6, 0.20% (all w/v). On the left is an identical SDS-PAGE gel containing marker proteins of the indicated molecular mass in kDa, stained with Coomassie Blue. The original scanned immunoblot images were uniformly altered electronically to increase the contrast and enhance the visibility of the weaker bands in the final photographic images.

 



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  		FIG. 5.
Effect of GSAT on GTR activity as measured by the rate of glutamyl-tRNA-dependent NADPH oxidation. A, the time course is shown for the change in A340 of unsupplemented assay mixture (diamonds) and assay mixtures supplemented with GTR alone (squares) or with GTR plus wild-type (triangles) or inactive K273A (circles) GSAT. B, the change in A340 is shown between 0 and 900 s for assay mixtures containing the indicated additions or omissions.

 



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  		FIG. 6.
Influence of GTR on GSAT activity. The control (100%) activity was 5.8 nmol of ALA formed in 30 min in 0.5-ml incubations containing 6 µg of wild-type GSAT. Each bar represents the mean and S.D. of the results of four experiments.

 
GTR Does Not Affect GSAT Activity—The ability of GSAT to stimulate GTR activity suggested the possibility that a complementary stimulation of GSAT activity by GTR might also occur. The conversion of GSA to ALA by GSAT was determined in the presence and absence of GTR or an unrelated protein, bovine serum albumin (BSA). Neither wild-type GTR, inactive C48R GTR, nor BSA had any effect on GSAT activity when present in assay mixtures in amounts equal to that of GSAT (Fig. 6).

Effect of Inactive K273A GSAT on ALA Formation from Glutamyl-tRNA—The rate of ALA production from glutamyl-tRNA was measured in a coupled enzyme assay containing approximately equimolar amounts of purified recombinant wild-type GTR and GSAT. Addition of inactive K273A GSAT to the assay mixture decreased the formation of ALA in a concentration-dependent manner (Fig. 7). In contrast, supplementation of the assay mixture with additional wild-type GSAT resulted in a small increase in ALA production. Addition of the unrelated protein BSA had no effect on ALA production.

If the decrease of ALA production from glutamyl-tRNA in the coupled enzyme assay is due to competition between wild-type and K273 GSAT for binding to GTR in a substrate-channeling scenario, then it would be expected that addition of increasing amounts of wild-type GSAT would reverse this inhibition. To test this hypothesis, excess wild-type GSAT was added to assay mixtures containing GTR and K273A GSAT. Increasing amounts of added wild-type GSAT caused increasing amounts of ALA formation. When wild-type GSAT was present at a concentration equal to twice that of K273A GSAT, ALA formation was restored to the level in assays containing wild-type GSAT and no K273A GSAT. The ability of wild-type GSAT to completely reverse the inhibitory effect of K273A GSAT is consistent with competition for binding sites on GTR and supports the hypothesis of substrate channeling of GSA between GTR and GSAT.



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  		FIG. 7.
Effects of inactive K273A GSAT on formation of ALA from glutamyl-tRNA. The control (100%) activity (bar 1) was 108 pmol ALA formed in 30 min in 0.1-ml incubations in microtiter plates containing 40 µg of GTR and 40 µg of GSAT. For bar 2, K273A GSAT was substituted for wild-type GSAT. For bar 3, an equal weight of BSA was substituted for GSAT. A, B, and C, excess wild-type GSAT, K273A GSAT, or BSA, respectively, were added to assay mixtures identical to that for bar 1. D, excess wild-type GSAT was added to assay mixtures identical to that for bar 2. A–D, the bars, reading from left to right, are for assays supplemented with the indicated component at 0.2, 0.5, 1.0, 1.2, and 1.5 times the weight of GSAT in the unsupplemented assay mixture. Each bar represents the mean and S.D. of the results of three experiments.

 
It is possible that the ability of catalytically inactive K273A GSAT to lower the formation of ALA in the coupled enzyme assay is due to secondary effects on the rate of ALA production. For example, K273A GSAT might sequester the intermediate GSA, lowering the concentration of free GSA available as a substrate for the wild-type GSAT, and thereby cause the observed decrease in ALA production. If sequestration of the intermediate were the cause of the decreased ALA production, the time course for ALA accumulation would be expected to exhibit a lag phase until enough free GSA accumulated to be available to wild-type GSAT, followed by a steady-state rate equal to that of control incubations. However, in incubations containing a constant amount of wild-type GSAT plus different amounts of K273A GSAT, the steady-state rate of ALA formation, measured at time points beginning after the first 5 min of the incubation, varied inversely with the amount of K273A GSAT in the incubations (Fig. 8).

Because GSAT is a native homodimer (15), another possible explanation for the ability of K273A GSAT to inhibit ALA formation from glutamyl-tRNA in the coupled enzyme assay is that wild-type and K273A GSAT might form inactive heterodimers that lower the overall GSAT activity and therefore lower ALA production. To test this possibility, the ability of wild-type GSAT to catalyze ALA formation from GSA in the presence of excess K273A GSAT was determined. Increasing amounts of K273A GSAT or the unrelated protein BSA had no effect on GSAT activity (Fig. 9). As expected, increasing amounts of wild-type GSAT caused an increase in ALA production from GSA. Also, addition of increasing amounts of wild-type GSAT to assay mixtures containing only K273A GSAT resulted in increasing amounts of ALA production. Therefore, inactive heterodimer formation is excluded as an explanation for the ability of K273A GSAT to inhibit ALA formation from glutamyl-tRNA in the coupled enzyme assay.

Another possible alternative explanation for the inhibitory effect of K273 GSAT is that it directly inhibits GTR. However, this explanation is excluded by results showing that GTR activity, as measured by NADPH oxidation, is stimulated rather than inhibited by GSAT whether the GSAT is active or inactive.



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  		FIG. 8.
Time course of the effect of inactive K273A GSAT on the conversion of glutamyl-tRNA to ALA. Incubations (30 min) contained 40 µg of wild-type GTR and 40 µg of wild-type GSAT in 0.1-ml volume in microtiter plates. Assay mixtures were supplemented with inactive K273A GSAT in the following amounts: none (circles), 20 µg (squares), 40 µg(diamonds), and 80 µg(triangles). The result at 30 min of an incubation containing 40 µg of wild-type GTR and 40 µg of K273A GSAT (but no wild-type GSAT) is also shown (X).

 



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  		FIG. 9.
Effect of inactive K273A GSAT on conversion of GSA to ALA by wild-type GSAT. The control (100%) activity (bar 1) was 3.6 nmol ALA formed in 30 min in 0.5-ml incubations containing 6 µg of wild-type GSAT. For bar 2, K273A GSAT was substituted for wild-type GSAT. For bar 3, an equal weight of BSA was substituted for GSAT. A–C, assay mixtures identical to that for bar 1 were supplemented with wild-type GSAT, K273A GSAT, or BSA, respectively. D, assay mixtures identical to that for bar 2 were supplemented with wild-type GSAT. A–D, the bars, reading from left to right, are for assays supplemented with the indicated component at 0.2, 0.5, 1.0, 1.2, and 1.5 times the weight of GSAT in the unsupplemented assay mixture.

 
Overall, these results clearly indicate that the ability of K273A GSAT to inhibit ALA formation from glutamate in the coupled enzyme assay is not due to inhibition of GTR activity. The results support the hypothesis that GSA is channeled between GTR and GSAT and that the ability of K273A GSAT to inhibit ALA formation from glutamyl-tRNA in the coupled enzyme assay is due to competition with wild-type GSAT for binding to GTR.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
This study presents physical and kinetic evidence for the interaction of GTR and GSAT and substrate channeling of the intermediate GSA in the formation of ALA from glutamyl-tRNA. Modeling studies based on the crystal structure of GTR from M. kandleri (13) and GSAT from Synechococcus sp. 6301 (16) had suggested the possibility of a complex between GTR, GSAT, and glutamyl-tRNA (13). In the model, dimeric GSAT docks within a V-shaped dimeric structure of GTR. The two enzymes can be aligned so that it is possible for the GSA intermediate to be channeled from the GTR active site to the active site of GSAT located ~25 Å away (13). Our results with C. reinhardtii GTR and GSAT support this hypothesis.

Co-immunoprecipitation and sucrose gradient ultracentrifugation results indicate the formation of a complex between GTR and GSAT. The interaction appears to be specific to the two proteins. The calculated molecular mass of the sucrose gradient sedimentation product is consistent with the formation of a 1:1 complex of native homodimeric GTR and homodimeric GSAT.

Glutaraldehyde cross-linking was used to examine whether GTR and GSAT form a complex in vivo. The results indicated that both proteins are present in a high molecular mass complex. The approximate molecular mass of the complex, ~250 kDa, is larger than the value that is expected from the in vitro sucrose gradient sedimentation results and is also larger than the calculated size of a 1:1 complex of homodimeric GTR and homodimeric GSAT, suggesting the possibility that the complex contains additional proteins. Two other proteins have been observed to interact with GTR. Earlier, glutamyl-tRNA synthetase was reported to form a complex with GTR in vitro in the presence of glutamyl-tRNA (26). An interaction between these two enzymes could be important in controlling the allocation of glutamyl-tRNA to the tetrapyrrole pathway by channeling this precursor, thus avoiding competition for the glutamyl-tRNA with the protein synthesis machinery. Recently, another protein, FLP, was reported to interact with GTR (23). FLP is a membrane-associated C. reinhardtii protein that is similar to the A. thaliana FLU protein, a negative regulator of GTR activity (24, 25). Using specific antibodies against barley glutamyl-tRNA synthetase and C. reinhardtii FLP, both proteins were detected on immunoblots of extracts from untreated cells. However, neither protein was detected in the high molecular mass complex that contained GTR and GSAT on immunoblots of extracts of glutaraldehyde-treated cells.

Protein-protein interactions allow for direct delivery of intermediates from an active site of one enzyme to an active site of another enzyme without the need for diffusion through the medium (28, 29). This phenomenon, called substrate channeling, has several potential advantages over free diffusion in solution. First, the transit time of intermediates is shorter when the enzymes are in a complex (30). Second, unstable intermediates are protected from the external environment (31). Third, intermediates can be segregated from competing enzymes. Protein-protein interactions and substrate channeling have been identified in many metabolic pathways including purine and pyrimidine synthesis, amino acid and lipid metabolism, glycolysis, the tricarboxylic acid cycle, DNA replication, RNA synthesis, and protein biosynthesis (32–36).

Substrate channeling of GSA between GTR and GSAT was investigated by kinetic studies of ALA production from glutamyl-tRNA in a coupled enzyme system. In a coupled two-enzyme system exhibiting substrate channeling, the presence of an inactive second enzyme would be predicted to lower the rate of conversion of the initial substrate to the final product by competing with active second enzyme for access to the intermediate residing on the first enzyme (37). Addition of inactive K273A GSAT to incubation mixtures containing GTR and wild-type GSAT decreased ALA formation from glutamyl-tRNA but not from GSA. Addition of like quantities of either wild-type GSAT or an unrelated protein, BSA, did not inhibit the coupled enzyme system. These results are most easily explained by competition between active and inactive GSAT molecules for docking sites on GTR and productive channeling of GSA only to active GSAT molecules. Alternative explanations, such as formation of inactive heterodimers between subunits of wild-type and K273A GSAT, or sequestration of GSA by inactive GSAT, were eliminated by results of experiments designed to test these possibilities.

Nearly complete inhibition of ALA production from glutamyl-tRNA was obtained at a 2-fold excess of inactive K273A GSAT over wild-type GSAT and GTR in the incubations. This result suggests that there is very little free diffusion of GSA under the in vitro assay conditions. Therefore, it can be concluded that GSA that is generated from glutamyl-tRNA by GTR proceeds to the GSAT active site primarily or solely through direct channeling when the two enzymes are present in an approximately equimolar ratio.

The crystal structure of GTR from M. kandleri indicates that the catalytic domain and the NADPH-binding domain are separated by 21 Å (13). Therefore, the NADPH-binding domain would have to move considerably to be in close proximity to reduce the substrate. It was proposed that such a dramatic change in GTR conformation could be possible upon glutamyl-tRNA·GTR·GSAT complex formation (13). We tested this hypothesis by measuring the rate of glutamyl-tRNA-dependent NADPH oxidation by GTR in the presence or absence of GSAT. This reaction is independent of the need for conversion of GSA to ALA. Nevertheless, GSAT greatly stimulated the reaction. Moreover, approximately equal stimulation was elicited by wild-type GSAT and inactive K273A GSAT, but not by the unrelated protein BSA. These results strongly support the conclusion that GTR and GSAT specifically interact and that the interaction activates GTR. Moreover, the ability of inactive GSAT to stimulate glutamyl-tRNA-dependent GTR activity indicates that GSAT-catalyzed conversion of GSA to ALA is not necessary for the release of the GSA product from GTR. Instead, the effect of the interaction between GTR and GSAT is to enhance the delivery to GSAT of GSA that would otherwise be released into the medium.

The physical and kinetic results presented here converge on the conclusion that GTR and GSAT function as a heteromolecular complex that activates GTR and facilitates channeling of the GTR product, GSA, to GSAT. This study did not identify additional proteins that might be present in a multienzyme complex along with GTR and GSAT to provide further channeling of intermediates. Nevertheless, the tetrapyrrole biosynthetic pathway, with its abundance of unstable and reactive intermediates, may yield other instances of substrate channeling. Indeed, in addition to the report of interaction between glutamyl-tRNA synthetase and GTR (26), there have been preliminary reports of results that are consistent with complex formation and substrate channeling between other tetrapyrrole biosynthetic enzymes, including porphobilinogen deaminase and uroporphyrinogen III synthase (38), protoporphyrinogen oxidase and ferrochelatase (39), and early steps of the Mg branch leading to chlorophylls and bacteriochlorophylls (40, 41). Further studies with purified enzymes operating under defined kinetic conditions will be necessary to test these possibilities.


   FOOTNOTES
 
* This work was supported by National Science Foundation Grant MCB-9808578 (to S. I. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Biomed. Box G-J4, Brown University, Providence, RI 02912. Tel.: 401-863-3129; Fax: 401-863-1182; E-mail: sib{at}brown.edu.

1 The abbreviations used are: ALA, 5-aminolevulinic acid; BSA, bovine serum albumen; GSA, glutamate 1-semialdehyde; GSAT, glutamate-1-semialdehyde aminotransferase; GTR, glutamyl-tRNA reductase; Ni-NTA, nickel-nitrilotriacetic acid. Back


   ACKNOWLEDGMENTS
 
We thank B. Grimm for antibody against barley chloroplast glutamyl-tRNA synthetase and J.-D. Rochaix for antibody against C. reinhardtii FLP antibody and for making a study available to us prior to publication.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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