RESUMEN
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.
Asunto(s)
Arabidopsis/metabolismo , Liasas de Carbono-Oxígeno/genética , Carbono/metabolismo , Ácido Fólico/metabolismo , Secuencia de Aminoácidos , Arabidopsis/efectos de los fármacos , Liasas de Carbono-Oxígeno/metabolismo , Liasas de Carbono-Azufre/genética , Liasas de Carbono-Azufre/metabolismo , Células Cultivadas , Antagonistas del Ácido Fólico/farmacología , Regulación de la Expresión Génica de las Plantas , Metionina/biosíntesis , Datos de Secuencia MolecularRESUMEN
Despite recent progress in elucidating the regulation of methionine (Met) synthesis, little is known about the catabolism of this amino acid in plants. In this article, we present several lines of evidence indicating that the cleavage of Met catalyzed by Met gamma-lyase is the first step in this process. First, we cloned an Arabidopsis cDNA coding a functional Met gamma-lyase (AtMGL), a cytosolic enzyme catalyzing the conversion of Met into methanethiol, alpha-ketobutyrate, and ammonia. AtMGL is present in all of the Arabidopsis organs and tissues analyzed, except in quiescent dry mature seeds, thus suggesting that AtMGL is involved in the regulation of Met homeostasis in various situations. Also, we demonstrated that the expression of AtMGL was induced in Arabidopsis cells in response to high Met levels, probably to bypass the elevated Km of the enzyme for Met. Second, [13C]-NMR profiling of Arabidopsis cells fed with [13C]Met allowed us to identify labeled S-adenosylmethionine, S-methylmethionine, S-methylcysteine (SMC), and isoleucine (Ile). The unexpected production of SMC and Ile was directly associated to the function of Met gamma-lyase. Indeed, we showed that part of the methanethiol produced during Met cleavage could react with an activated form of serine to produce SMC. The second product of Met cleavage, alpha-ketobutyrate, entered the pathway of Ile synthesis in plastids. Together, these data indicate that Met catabolism in Arabidopsis cells is initiated by a gamma-cleavage process and can result in the formation of the essential amino acid Ile and a potential storage form for sulfide or methyl groups, SMC.
Asunto(s)
Arabidopsis/metabolismo , Liasas de Carbono-Azufre/metabolismo , Cisteína/análogos & derivados , Isoleucina/biosíntesis , Metionina/metabolismo , Alquinos/metabolismo , Arabidopsis/citología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Liasas de Carbono-Azufre/genética , Cisteína/biosíntesis , Glicina/análogos & derivados , Glicina/metabolismo , Resonancia Magnética Nuclear Biomolecular , Compuestos de Sulfonilurea/metabolismoRESUMEN
The subcellular distribution of Met and S-adenosylmethionine (AdoMet) metabolism in plant cells discloses a complex partition between the cytosol and the organelles. In the present work we show that Arabidopsis contains three functional isoforms of vitamin B(12)-independent methionine synthase (MS), the enzyme that catalyzes the methylation of homocysteine to Met with 5-methyltetrahydrofolate as methyl group donor. One MS isoform is present in chloroplasts and is most likely required to methylate homocysteine that is synthesized de novo in this compartment. Thus, chloroplasts are autonomous and are the unique site for de novo Met synthesis in plant cells. The additional MS isoforms are present in the cytosol and are most probably involved in the regeneration of Met from homocysteine produced in the course of the activated methyl cycle. Although Met synthesis can occur in chloroplasts, there is no evidence that AdoMet is synthesized anywhere but the cytosol. In accordance with this proposal, we show that AdoMet is transported into chloroplasts by a carrier-mediated facilitated diffusion process. This carrier is able to catalyze the uniport uptake of AdoMet into chloroplasts as well as the exchange between cytosolic AdoMet and chloroplastic AdoMet or S-adenosylhomocysteine. The obvious function for the carrier is to sustain methylation reactions and other AdoMet-dependent functions in chloroplasts and probably to remove S-adenosylhomocysteine generated in the stroma by methyltransferase activities. Therefore, the chloroplastic AdoMet carrier serves as a link between cytosolic and chloroplastic one-carbon metabolism.
Asunto(s)
Cloroplastos/metabolismo , Citosol/metabolismo , Metionina/química , Metionina/metabolismo , S-Adenosilmetionina/química , Arabidopsis/metabolismo , Western Blotting , Clonación Molecular , ADN Complementario/metabolismo , Difusión , Relación Dosis-Respuesta a Droga , Escherichia coli/metabolismo , Proteínas Fluorescentes Verdes , Homocisteína/química , Immunoblotting , Cinética , Proteínas Luminiscentes/metabolismo , Mitocondrias/metabolismo , Modelos Biológicos , Datos de Secuencia Molecular , Pisum sativum , Filogenia , Plastidios/metabolismo , Isoformas de Proteínas , Factores de Tiempo , Vitamina B 12/metabolismoRESUMEN
Tetrahydrofolate (THF) is a central cofactor for one-carbon transfer reactions in all living organisms. In this study, we analyzed the expression of dihydropterin pyrophosphokinase-dihydropteroate synthase (HPPK-DHPS) in pea (Pisum sativum) organs during development, and so the capacity to synthesize dihydropteroate, an intermediate in the de novo THF biosynthetic pathway. During seedling development, all of the examined organs/tissues contain THF coenzymes, collectively termed folate, and express the HPPK-DHPS enzyme. This suggests that each organ/tissue is autonomous for the synthesis of THF. During germination, folate accumulates in cotyledons and embryos, but high amounts of HPPK-DHPS are only observed in embryos. During organ differentiation, folate is synthesized preferentially in highly dividing tissues and in photosynthetic leaves. This is associated with high levels of the HPPK-DHPS mRNA and protein, and a pool of folate 3- to 5-fold higher than in the rest of the plant. In germinating embryos and in meristematic tissues, the high capacity to synthesize and accumulate folate correlates with the general resumption of cell metabolism and the high requirement for nucleotide synthesis, major cellular processes involving folate coenzymes. The particular status of folate synthesis in leaves is related to light. Thus, when illuminated, etiolated leaves gradually accumulate the HPPK-DHPS enzyme and folate. This suggests that folate synthesis plays an important role in the transition from heterotrophic to photoautotrophic growth. Analysis of the intracellular distribution of folate in green and etiolated leaves indicates that the coenzymes accumulate mainly in the cytosol, where they can supply the high demand for methyl groups.