Tomato gamma-glutamylhydrolases: expression, characterization, and evidence for heterodimer formation

Abstract

Folates typically have gamma-linked polyglutamyl tails that make them better enzyme substrates and worse transport substrates than the unglutamylated forms. The tail can be shortened or removed by the vacuolar enzyme gamma-glutamyl hydrolase (GGH). It is known that GGH is active only as a dimer and that plants can have several GGH genes whose homodimeric products differ functionally. However, it is not known whether GGH dimers dissociate under in vivo conditions, whether heterodimers form, or how heterodimerization impacts enzyme activity. These issues were explored using the GGH system of tomato (Solanum lycopersicum). Tomato has three GGH genes that, like those in other eudicots, apparently diverged recently. LeGGH1 and LeGGH2 are expressed in fruit and all other organs, whereas LeGGH3 is expressed mainly in flower buds. LeGGH1 and LeGGH2 homodimers differ in bond cleavage preference; the LeGGH3 homodimer is catalytically inactive. Homodimers did not dissociate in physiological conditions. When coexpressed in Escherichia coli, LeGGH1 and LeGGH2 formed heterodimers with an intermediate bond cleavage preference, whereas LeGGH3 formed heterodimers with LeGGH1 or LeGGH2 that had one-half the activity of the matching homodimer. E. coli cells expressing LeGGH2 showed approximately 85% reduction in folate polyglutamates, but cells expressing LeGGH3 did not, confirming that LeGGH2 can function in vivo and LeGGH3 cannot. The formation of LeGGH1-LeGGH2 heterodimers was demonstrated in planta using bimolecular fluorescence complementation. Plant GGH heterodimers thus appear to form wherever different GGH genes are expressed simultaneously and to have catalytic characteristics midway between those of the corresponding homodimers.

Figure 1.

Structure of tetrahydrofolate polyglutamates. One-carbon units at various oxidation levels can be coupled to the N⁵ and/or N¹⁰ position. A γ-linked polyglutamyl tail of up to about six residues is attached to the first Glu. Folates undergo oxidative cleavage of the C⁹-N¹⁰ bond yielding pterin and pABAGlu_n moieties. The arrows show bonds cleaved by GGH.

Figure 2.

Southern-blot analysis and GGH phylogeny. A, Tomato genomic DNA (10 μg) was digested with restriction enzyme BstnI, DraI, EcoRI, EcoRV, HaeIII, or ScaI. The digested samples were separated on a 1% agarose gel, blotted to nitrocellulose, and hybridized to gene-specific ³²P-labeled probes corresponding to the 3′-UTR of each GGH gene (top two frames and bottom left frame) or to the coding region of LeGGH2 (bottom right frame). B, Evolutionary relationships of GGH sequences of tomato (Le), Arabidopsis (At), soybean (Gm), Populus trichocarpa (Pt), rice (Os), barley (Hv), and maize (Zm). Sequences were extracted from genome and EST databases. The tree was constructed by the neighbor-joining method. The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to branches. The tree is drawn to scale; evolutionary distances are in units of the number of amino acid substitutions per site.

Figure 3.

LeGGH gene expression and enzyme activity. A, Northern analysis. Total RNA (15 μg) was fractionated on a 1% agarose-formaldehyde gel, blotted to a nylon membrane, and hybridized to gene-specific ³²P-labeled probes corresponding to the 3′-UTR of each gene. B, GGH activity was assayed in desalted crude tissue extracts using 0.2 mm PteGlu₅ as substrate. Specific activity values are the means ± se of three independent experiments using tissue pooled from three to five different plants. MG, Mature green fruit; Br, breaker stage fruit; RR, red ripe fruit; L, leaf; R, root; S, stem; Sh, shoot; P, pedicel; FB, flower bud; F, flower; Se, sepal; Pe, petal; St, stamen; C, carpel.

Figure 4.

Recombinant GGH purification, bond cleavage specificity, and in vivo activity. A, Purification of His₆-tagged LeGGH1 and LeGGH2 homodimers by Ni²⁺ affinity chromatography; a subsequent cation exchange step was used for LeGGH1. Proteins were separated by SDS-PAGE and stained with Coomassie Blue. For each protein, lane 1 was loaded with E. coli extract containing 10 μg of total protein, and lane 2 with 2 μg of purified protein. B, Progress curves for hydrolysis of PteGlu₅ (0.1 mm) by LeGGH1 and LeGGH2. Data are presented as plots of relative concentration of each reaction product versus extent of reaction and are representative of results obtained in three independent experiments. Symbols and their numbers illustrate the number of Glu residues remaining on the pteroyl moiety following hydrolysis of the polyglutamate tail. C, Progress curves for hydrolysis of pABAGlu₅ (0.1 mm) by LeGGH1 and LeGGH2. Data plots are as above. D, Effect of LeGGH2 or LeGGH3 expression on folate polyglutamylation in E. coli. Bars show the extent of polyglutamylation of folates extracted from logarithmically growing E. coli cultures (A₆₀₀ = 0.5–0.7) harboring pET28b alone or containing LeGGH2 or LeGGH3. Folates were analyzed by HPLC with electrochemical detection; the species were tetrahydrofolate (THF), 5-methyltetrahydrofolate (5-CH₃-THF), 5,10-methenyltetrahydrofolate (5,10-CH-THF), and 5-formyltetrahydrofolate (5-CHO-THF). For each species, the data show percentages of total folate that were polyglutamylated (two to eight Glu residues) or were in the monoglutamyl form. Data are mean values from three independent experiments.

Figure 5.

Conservation of the GGH dimer interface and evidence for formation of nondissociating LeGGH homo- and heterodimers. A, Conservation of the dimer interface. Amino acids that form the dimer interface comprise two helices (α2 and α9) and a single β-strand (β13). Numbers correspond to those amino acids forming the interface and are based on the human GGH crystal structure (Li et al., 2002). B, Dimerization. LeGGH monomers carrying His₆ or FLAG tags were expressed in E. coli singly or in pairs; extracted proteins were then analyzed by western blotting using antibodies to the His₆ tag, before (bottom) and after (top) affinity purification on FLAG M2 resin. When differentially tagged LeGGH2 proteins were coexpressed, homodimers formed, as shown by detection of the His₆ epitope in FLAG-affinity-purified samples. When LeGGH1-FLAG or LeGGH3-FLAG was coexpressed with LeGGH2-His₆, or LeGGH1-FLAG with LeGGH3-His₆, heterodimer formation was evident from the presence of the His₆ epitope in FLAG-affinity-purified proteins. C, Dimer stability. Equimolar amounts of LeGGH2-FLAG and LeGGH3-His₆ homodimers were incubated together at 30°C for up to 4 h in physiological conditions (100 mm potassium phosphate, pH 6.0, 10% glycerol). At intervals, samples were applied to FLAG M2 resin, and the bound fraction was analyzed by western blotting using antibodies to the His₆ tag. LeGGH2-His₆/LeGGH3-FLAG purified heterodimer standards corresponding to 2% to 10% heterodimer formation were treated the same way. A portion of the homodimer mixture (Input) was analyzed prior to the affinity step as a positive control. The experimental sample blot was stained with Ponceau red to confirm protein recovery from the affinity resin (bottom).

Figure 6.

The impact of heterodimer formation on GGH activity and bond cleavage specificity and evaluation of folate binding by LeGGH3. A, GGH activity. Doubly His₆- and FLAG-tagged homodimers of LeGGH2 (2-2) and heterodimers of LeGGH1 and LeGGH2 (1-2), LeGGH2 and LeGGH3 (2-3), or LeGGH1 and LeGGH3 (1-3) were affinity purified and tested for total GGH activity. Data are means and se for three experiments. The purity of the purified proteins was verified by SDS-PAGE and staining with Coomassie Blue (inset). Note that the LeGGH1, LeGGH2, and LeGGH3 proteins are resolved from each other. B, Products formed from PteGlu₅ by GGH homo- and heterodimers. Homo- and heterodimers were as above, with the addition of the LeGGH1 homodimer (1-1). C, Effect on GGH activity and cleavage product profile of adding the catalytically inactive LeGGH3 homodimer (0–10 μm) to reaction mixtures containing 1 μm PteGlu₅ and 5 ng of LeGGH2 homodimer.

Figure 7.

BiFC evidence for GGH heterodimer formation in planta. A, Representative fluorescence (i) and bright-field (ii) images of Arabidopsis protoplasts transiently expressing YFP, its N-terminal (YFP_N) and C-terminal (YFP_C) fragments, or YFP_N and YFP_C as fusions to LeGGH1, LeGGH2, and/or the signal peptide of LeGGH1 (sp1), as indicated on the right of the images. B, Percentage of the transformed protoplasts above exhibiting BiFC signals from a minimum of three independent experiments. BiFC intensity was visually scored as low, medium, or high (inset) from a minimum of 100 protoplasts viewed for each transformation. Note the lack of high intensity BiFC signals from protoplasts transformed with YFP_N/YFP_C or GGH1-YFP_N/sp1-YFP_C combinations.