Cloning and characterisation of a pea mitochondrial NDPK.
Here we report the cloning of a cDNA encoding the first nucleoside diphosphate kinase (NDPK) isolated from plant mitochondria. Amplification of a 317 nt product was performed by PCR, using oligonucleotide primers based on partial amino acid sequences of the pea mitochondria NDPK and other NDPK isoforms. By screening of a pea cDNA library with this PCR product, a full length clone was obtained. Northern analysis revealed the presence of a 1.1 kb single transcript, with high expression in young leaves and reproductive tissues. The clone encodes a precursor protein of 232 amino acids (26 kDa), including an N-terminal extension of 80 amino acids (9 kDa). Analysis of the deduced amino acid sequence confirmed its identity with the sequences obtained from the purified mature pea mitochondrial NDPK. In vitro import experiments carried out in isolated pea mitochondria showed targeting and processing of the 27 kDa precursor into a 16.5 kDa mature form. Phylogenetic analysis of some vertebrate and plant isoforms of NDPK showed that the pea mtNDPK groups together with the NDPK3 isoform from A. thaliana and the chloroplastic NDPK III from spinach. We suggest that it is possible to design a novel classification of the different NDPK isoforms according to their subcellular localisation and origin.
Escobar Galvis
M L
ML
Plant Cell Biology, Lund University, Sweden.
Håkansson
G
G
Alexciev
K
K
Knorpp
C
C
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D018744:DNA, Plant / Q000235:genetics / Q000302:isolation & purification; D015870:Gene Expression; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D009701:Nucleoside-Diphosphate Kinase / Q000235:genetics*; D018532:Pisum sativum / Q000201:enzymology* / Q000235:genetics*; D010802:Phylogeny; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D018076:DNA, Complementary; D018744:DNA, Plant; D009701:Nucleoside-Diphosphate Kinase