Chloroplasts methionine synthesis

In pea seedlings during germination there is a massive synthesis of homoserine, an intermediate in threonine and methionine synthesis (see Bryan, this volume. Chapter 11). There is little doubt that homoserine is derived from aspartate in pea chloroplasts (Lea et aL, 1979a) but whether this is the sole route of homoserine biosynthesis in pea seedlings is still in doubt (Mitchell and Bidwell, 1970 Bauer ef a/., 1977b). 

An alternative approach to estimating the metabolic capabilities of chloroplasts entails measurement of the light-dependent metabolism of radioactive tracers. Using isolated pea chloroplasts. Mills and Wilson (1978a) found that lysine, methionine, threonine, and isoleucine were synthesized from [ C]aspartate. Further evidence that aspartate was being metabolized via the anticipated pathways was provided by the demonstration that the synthesis of homoserine was inhibited by lysine and threonine (Lea et al., 1979). These results, combined with those relating to enzyme localization, lead to the concept that chloroplasts contain a complete functional sequence of enzymes which can facilitate the synthesis of the aspartate family and at least some of the branched-chain amino acids. This is consistent with the importance of chloroplasts in ammonia assimilation (Miflin and Lea, this volume. Chapter 4) and with the evidence that protein can be synthesized from CO2 in isolated plastids (Shepard and Leven, 1972 Huberer al., 1977). The actual fraction of [ ]02 which is utilized for amino acid biosynthesis in isolated plastids is usually quite small. Thus, reactions which normally occur outside of chloroplasts are considered to be of major importance in the synthesis of carbon skeletons such as oxaloacetate or pyruvate (Kirk and Leech, 1972 Leech and Murphy, 1976). 

Plants, in common with microorganisms and animals, require methionine chiefly for three roles, (a) As a component of protein, a role which accounts for most of the methionine in the cell, (b) As a component of methionyl tRNA (in eukaryotes) and formylmethionyl tRNA (in chloroplasts, mitochondria, and prokaryotes), factors required for initiation of protein synthesis. (c) As a component of AdoMet, the chief biological methyl donor, the obligatory precursor of spermidine and spermine, and an effector of certain enzymes. In addition to these chief roles, a major pathway for the metabolism of methionine in certain plant tissues is its conversion to ethylene (see Yang and Adams, this series, Vol. 4, Chapter 6). Only plants and microorganisms can synthesize the homocysteine moiety of methionine novo, and the importance of this synthesis in the sulfur cycle has been noted in the introduction. 

These combined findings indicate that chloroplasts catalyze the conversion of aspartate to cystathionine, although it is not known to what extent these reactions may also be catalyzed by extrachloroplastic fractions. The subcellular site(s) for further metabolism by cystathionine-j8-Iyase, tetrahy-dropteroyltriglutamate methyltransferase and methionine adenosyltransferase is not clear, although an appreciable percentage of the total tetrahydro-pteroyltriglutamate methyltransferase can reside in mitochondria. Further studies are obviously required to establish the quantitative interrelationship of chloroplasts and other subcellular sites in the de novo-synthesis of methionine and AdoMet. 

Fig. 14. Comparison of products of protein synthesis by intact and lysed chloroplasts. Chloro-plasts were isolated as in Fig. 1, and an aliquot was lysed by resuspension in 25 mM Tricine-KOH, 10 mM MgS04, 5 mM 2-mercaptoethanol pH 8.0). Intact chloroplasts were incubated with p S] methionine and light as energy source, as in Fig. 1. Lysed chloroplasts were incubated with p S]methionine, 100 mM KCl, 2 mM ATP and 0.2 mM GTP in the lysing medium. Intact (----) and lysed (---) chloroplasts were analyzed on 15% SDS gels.

Fig. 15. SDS gel electrophoresis of soluble products of protein synthesis by free chloroplast ribosomes. Chloroplasts were lysed as in Fig. 14, and the lysate was centrifuged at 38,OOOg for 5 min. The colorless supernatant was incubated with [ S]methionine and ATP and GTP for 40 min, and then centrifuged at 200,000g for 3 h. The supernatant fraction was analyzed on 15% SDS gels. Symbols as in Fig. 3.

A further factor to consider with respect to the localization of homocysteine synthesis in chloroplasts is whether the intermediates of the pathways involved are present at concentrations which sustain homocysteine synthesis at rates commensurate with the rate of production of methionine by the whole cell or organism. In this regard Giovanelli et al. (1986) reported that cystathionine... 

Information is accumulating concerning the mechanism of initiation of protein synthesis. In bacterial, mitochondrial and chloroplast systems the initial amino acid in a polypeptide is always formyl-methionine—the formyl residue being removed from the completed... 

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