Regulation of one-carbon metabolism in Arabidopsis: the N-terminal regulatory domain of cystathionine gamma-synthase is cleaved in response to folate starvation

Abstract

In all organisms, control of folate homeostasis is of vital importance to sustain the demand for one-carbon (C1) units that are essential in major metabolic pathways. In this study we induced folate deficiency in Arabidopsis (Arabidopsis thaliana) cells by using two antifolate inhibitors. This treatment triggered a rapid and important decrease in the pool of folates with significant modification in the distribution of C1-substituted folate coenzymes, suggesting an adaptive response to favor a preferential shuttling of the flux of C1 units to the synthesis of nucleotides over the synthesis of methionine (Met). Metabolic profiling of folate-deficient cells indicated important perturbation of the activated methyl cycle because of the impairment of Met synthases that are deprived of their substrate 5-methyl-tetrahydrofolate. Intriguingly, S-adenosyl-Met and Met pools declined during the initial period of folate starvation but were further restored to typical levels. Reestablishment of Met and S-adenosyl-Met homeostasis was concomitant with a previously unknown posttranslational modification that consists in the removal of 92 amino acids at the N terminus of cystathionine gamma-synthase (CGS), the first specific enzyme for Met synthesis. Rescue experiments and analysis of different stresses indicated that CGS processing is specifically associated with perturbation of the folates pool. Also, CGS processing involves chloroplastic serine-type proteases that are expressed in various plant species subjected to folate starvation. We suggest that a metabolic effector, to date unidentified, can modulate CGS activity in vivo through an interaction with the N-terminal domain of the enzyme and that removal of this domain can suppress this regulation.

Figure 1.

Synthetic overview of C1 metabolism in plant cells. The synthesis of THF is shown on a gray background. The enzymes that are specifically inhibited by sulfanilamide and MTX are dihydropteroate synthase (1) and dihydrofolate reductase (2), respectively. The enzymes that utilize folate coenzymes are Ser hydroxymethyltransferase (3), Gly decarboxylase (4), and Met synthase (5). CGS (6) and cystathionine β-lyase (7) are involved in de novo synthesis of Met. AdoHcy hydrolase (8) catalyzes the reversible conversion of AdoHcy to Hcy and adenosine. Thr synthase (9) competes with CGS for the common substrate OPH to synthesize Thr. C1 units transported by THF are 10-formyl (10-CHO), 5-formyl (5-CHO), 5,10-methenyl (5,10-CH⁺), 5,10-methylene (5,10-CH₂), and 5-methyl (5-CH₃). cysta, Cystathionine.

Figure 2.

Analysis of folates pools in control and MTXS-treated Arabidopsis cells. Cells were grown in standard conditions or exposed to MTX (100 μm) and sulfanilamide (100 μm) over a 72 h period and folates were analyzed by LC-MS/MS (Zhang et al., 2005). Three pools of folates were considered: 5-methyl-THF, THF and 5,10-methylene-THF, and other derivatives (5-CHO-THF, 10-CHO-THF, and 5,10-methenyl-THF). Data are means of three to six biological replicates and sd. Note that the scale used for folates quantification is different for control and MTXS-treated cells.

Figure 3.

Analysis of key metabolites of C1 metabolism in folate-sufficient and folate-deficient Arabidopsis cells. Free amino acids, thiols, and AdoMet/AdoHcy were extracted from control (▪) and MTX-treated (○) cells collected at different time intervals and analyzed by LC as described in “Materials and Methods.” Data are means ± sd of three biological replicates.

Figure 4.

Expression of the enzymes involved in Met synthesis and catabolism in folate-sufficient and folate-deficient cells. Soluble proteins (40 μg per lane) from Arabidopsis cells grown in standard medium (control cells) or exposed to 100 μm MTX and 100 μm sulfanilamide (MTXS cells) were analyzed by western blot using antibodies raised against CGS, cystathionine β-lyase, Met synthases, and Met γ-lyase from Arabidopsis. The 53- and 50-kD polypeptides detected with the CGS serum are characteristic of the mature enzyme, the one at 43 kD being observed only in cells starved for folates for 72 h. Quantitation of CGS polypeptides using chemiluminescence detection reagents and a Typhoon 9400 scanner indicated that the amount of CGS protein was increased by 2-fold between 24 and 48 h of treatment and was maintained constant between 48 and 72 h (titration experiments using recombinant proteins indicated that the signal obtained with the 43-kD polypeptide of CGS was reduced by 30%–40% as compared with the signal measured for the mature protein). The antibodies against Met synthase cross-react with both the cytosolic (top band) and chloroplastic (bottom band) isoforms of the enzyme (Ravanel et al., 2004a).

Figure 5.

CGS activity in control and MTXS-treated Arabidopsis cells. Soluble proteins were extracted from control (gray bars) and MTX-treated (white bars) cells collected at different time intervals, desalted through Sephadex G25, and CGS activity was determined by LC after derivatization of cystathionine. Data are means ± sd of three biological replicates.

Figure 6.

Evidence for posttranslational cleavage of the N-terminal region of MCGS in folate-deficient cells. A, Soluble proteins prepared from control cells were mixed with an equal amount of proteins from cells treated with MTXS for 72 h (MTXS72) and incubated for 2 h at 25°C. Forty micrograms of proteins were analyzed in each lane. B, Pure recombinant CGS enzymes (25 ng) were incubated for 2 h at 25°C with 2.5 μg soluble proteins from MTXS72 cells. Two versions of the enzyme were analyzed: the MCGS (starting with Val-69) and a truncated CGS (TrCGS, starting with Ala-113) bearing a deletion of 44 residues at the N terminus of the protein. Each combination from sections A and B was analyzed by western blot with polyclonal antibodies raised against CGS. Note that in B only the recombinant CGSs are detected because they are present in excess as compared to the enzyme provided by the MTXS72 extract.

Figure 7.

Alignment of amino acid sequences of the N-terminal region of CGS from various plant species. The alignment was generated with ClustalW using the amino acid sequences of CGS from Arabidopsis (GenBank accession no. ATU83500), soybean (Glycine max; AAD34548), Medicago truncatula (ABE79443), tobacco (AB035300 and AF097180), and maize (AAB61347). Only the N-terminal region of CGSs is shown in the alignment. The mature (MCGS) and cleaved (CCGS) versions of the Arabidopsis enzyme start with residues Val-69 and Ser-161, respectively. The MTO1 (Goto et al., 2005) and catalytic (Steegborn et al., 1999) domains are underlined. The truncated Arabidopsis CGS enzyme overexpressed in transgenic tobacco plants by Hacham et al. (2002) starts with Ser-173 (asterisk).

Figure 8.

Processing of the N-terminal domain of CGS is specifically associated with folates starvation and occurs in several plant species. A, Soluble protein extracts were prepared from Arabidopsis cells grown for 72 h in culture medium supplemented with the following compounds, alone or in combination: 100 μm MTX, 100 μm sulfanilamide, 0.5 mm 5-formyl-THF, and 1 mm Met. Soluble proteins (30 μg) were extracted and analyzed by western blot with polyclonal antibodies against CGS. B, Leaf discs from pea and maize were floated for 8 d in petri dishes containing control medium or supplied with both MTX (100 μm) and sulfanilamide (100 μm). Soluble proteins (5 μg) prepared from control or MTXS-treated leaf discs were combined with an Arabidopsis protein extract (20 μg) containing MCGS and incubated for 1 h at 25°C before performing immunoblot analysis.