Isolation and sequence analysis of cDNAs encoding phosphoenolpyruvate carboxykinase from the PCK-type C4 grass Urochloa panicoides

Abstract

A rabbit antiserum was raised against phosphoenolpyruvate carboxykinase (PCK) purified from Urochloa panicoides, a PCK-type C4 monocot. The antiserum was used to screen a cDNA expression library constructed from U. panicoides leaf poly(A)+RNA. Inserts from immunoreactive clones were used to rescreen the library and obtain three overlapping cDNAs comprising a 2220 bp composite sequence. The single complete open reading frame of 1872 bp encodes PCK1, a 624 amino acid polypeptide with a predicted molecular mass of 68,474 Da. Comparison of PCK1 with other ATP-dependent PCKs indicates that PCK1 is significantly larger, mainly due to an N-terminal extension of greater than 65 residues, and reveals high sequence identity across the central portion of the protein, especially over seven sub-sequences. One of these sub-sequences spans motifs common to several ATP-utilising enzymes for phosphate and divalent cation binding. The anti-PCK antiserum recognises a 69 kDa polypeptide on immunoblots of either purified PCK or U. panicoides leaf extracts. However, polypeptides of 63, 62, 61 and 60 kDa are also immunoreactive. Amino terminal sequencing of polypeptides from preparations of purified PCK demonstrates that these smaller polypeptides are related to PCK1, and time course experiments show that these polypeptides arise from the breakdown of PCK during isolation. Northern blot analysis indicates that the 2.7 kb PCK mRNA is abundant in green leaves but not in roots or etiolated shoots. Moreover, PCK mRNA levels increase gradually during greening, reaching maximum levels after about 84 h.