Glycine decarboxylase complex from higher plants. Molecular cloning, tissue distribution and mass spectrometry analyses of the T protein.
cDNA clones encoding the precursor of the T protein of the glycine decarboxylase complex have been isolated from a pea leaf cDNA library in lambda gt11. 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 +/- 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 predicted 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 (H4PteGlun), either in the native T protein or in the different peptide fragments generated by the chemical cleavage. The absence of H4PteGlun bound to the T protein in our experimental conditions demonstrates that H4PteGlun 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 (approximately 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. Western blot analysis showed that the P, L and T proteins of the glycine decarboxylase complex were present in roots whereas the H protein was not 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.
Bourguignon
J
J
URA CNRS 576, Département de Biologie Moléculaire et Structurale, Grenoble, France.
Vauclare
P
P
Merand
V
V
Forest
E
E
Neuburger
M
M
Douce
R
R
D000595:Amino Acid Sequence; D050960:Aminomethyltransferase; D001483:Base Sequence; D015139:Blotting, Southern; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003488:Cyanogen Bromide; D018076:DNA, Complementary / Q000737:chemistry / Q000302:isolation & purification; D007887:Fabaceae / Q000235:genetics; D019877:Hydroxymethyl and Formyl Transferases; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010944:Plants / Q000201:enzymology*; D010946:Plants, Medicinal; D011487:Protein Conformation; D012333:RNA, Messenger / Q000032:analysis; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution; D014166:Transferases / Q000032:analysis / Q000737:chemistry / Q000235:genetics
D018076:DNA, Complementary; D012333:RNA, Messenger; D014166:Transferases; D019877:Hydroxymethyl and Formyl Transferases; D050960:Aminomethyltransferase; D003488:Cyanogen Bromide