Glycine decarboxylase complex from higher plants

Abstract

-EJB 93 0737/1 cDNA clones encoding the precursor of the T protein of the glycine decarboxylase complex have been isolated from a pea leaf cDNA library in 2 gtl 1. The longest cDNA insert of 1430 bp encodes a polypeptide of 408 amino acid residues of which 30 residues constitute an N-terminal cleavable presequence and 378 residues make up the mature protein. Several results confirmed the identity of the cDNA and the exactness of the predicted primary structure. Firstly, we purified the T protein to homogeneity and its mass was measured by mass spectrometry. The mass obtained (40966 i 5 Da) was the value predicted from the cDNA (40961 Da). Secondly, the purified T protein was chemically cleaved with cyanogen bromide and the peptide fragments were analysed by high-performance liquid chromatography/electrospray ionization mass spectrometry and/or fast-atom-bombardment mass spectrometry. The mass values of all the peptides generated by chemical cleavage and measured by these techniques were very close to the values calculated from the prcdictcd primary structure. Thirdly, microsequencing of some of these peptides, which represent 35% of the total protein, fits perfectly with the primary structure deduced from the cDNA. In the present HPLC/electrospray ionization MS studies we never detected the presence of covalently bound tetrahydropteroylpolyglutamate (H,PteGlu,,), either in the native T protein or in thc different pcptide fragments generalcd by the chemical cleavage. The absence of H,PteGlu,, bound to the T protein in our experimental conditions demonstrates that H,PteGlu, is not covalently linked to the T protein. Northern blot analysis showed that the steady-state level of the mRNA corresponding to the T protein was high in green leaves compared to the level in etiolated leaves (= 8-10-fold higher). Surprisingly, a non-negligible amount of mRNA corresponding to the T protein was present in roots whereas the mRNA encoding the H protein was not detectable. Westcrn blot analysis showed that the P, L and T proteins of the glycine decarboxylase complex were present in roots whereas the H protein was no1 detectable. Southern hybridization to pea genomic DNA indicated the presence of a single gene encoding the T protein of the glycine decarboxylase complex in the haploid genome.

Distribution of Folate Derivatives and Enzymes for Synthesis of 10-Formyltetrahydrofolate in Cytosolic and Mitochondrial Fractions of Pea Leaves

“…Second, the crossreacting 40-and 44-kD proteins may have some structural similarity to SYN but do not have this catalytic activity. In this regard, leaf mitochondria contain significant amounts of T-protein (subunit M, of approximately 41,000) as part of the GDC complex (Bourguignon et al, 1993). T-protein also binds tetrahydrofolate and has some structural similarity to the SYN domains of mammalian C,-tetrahydrofolate synthases (Kopriva et al, 1995).…”

Regulation of the Expression of the Glycine Decarboxylase Complex during Pea Leaf Development

“…The cDNAs encoding the H-, L-, and T-proteins that were isolated by Macherel et al (1990) and Bourguignon et al (1992Bourguignon et al ( , 1993 were used as probes for northern blot analysis. cDNA probes encoding the P-protein and rbcS 3A were amplified by PCR from a pea cDNA library in A gtll (Macherel et al, 1990) using specific oligonucleotides designed from the sequence of the corresponding cDNAs published by Turner et al (1992a) and Coruzzi et al (1983).…”

“…In plants P-, H-, T-, and L-proteins are synthesized from nuclear-encoded genes (Walker and Oliver, 1986b), which have been mapped on the pea (Pisum sativum) chromosomes (Turner et al, 1993). AI1 of the cDNAs corresponding to these proteins have been isolated and characterized (Kim and Oliver, 1990;Macherel et al, 1990;Bourguignon et al, 1992Bourguignon et al, , 1993Turner et al, 1992aTurner et al, , 1992b. Mitochondria isolated from etiolated pea leaves oxidized Gly at very low rates, whereas mitochondria from green leaves exhibited high rates of Gly oxidation and this was correlated to the presente in large amounts of the proteins of GDC (Day et al, 1985;Walker and Oliver, 1986b).…”

“…Mitochondria isolated from etiolated pea leaves oxidized Gly at very low rates, whereas mitochondria from green leaves exhibited high rates of Gly oxidation and this was correlated to the presente in large amounts of the proteins of GDC (Day et al, 1985;Walker and Oliver, 1986b). Northern and western blot analyses of the expression of the GDC proteins in pea revealed that the P-, H-, and T-proteins were expressed predominantly in the leaf tissue (Kim and Oliver, 1990;Macherel et al, 1990;Kim et al, 1991;Turner et al, 1992a;Bourguignon et al, 1993). However, in contrast to these three proteins, the expression of the L-protein occurs in a11 of the tissue examined so far.…”

Separation and identification of soybean leaf proteins by two-dimensional gel electrophoresis and mass spectrometry

“…It is a key enzyme in the biosynthesis of purines, lipids, hormones and other compounds (Kopriva and Bauwe, 1995). The glycine cleavage system catalyzes the degradation of glycine and is composed of four proteins: P, T, L and H proteins (Bourguignon et al, 1993). Other identified proteins belong in the protein destination and storage category: chaperonin (spot 95, 96, 100 and 113), HSP/HSC (heat shock protein/heat shock cognate) 70 and associated co-chaperones (spot 97, 98, 99, and 101), stem 28 kDa protein (spot 90-93), cyclophilin (spot 76, 102, and 103), endopeptidase Clp (spot 105) and polyubiquitin (spot 104).…”