JXB Advance Access originally published online on February 27, 2004
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Journal of Experimental Botany, Vol. 55, No. 398, pp. 955-956, April 1, 2004
© 2004 Oxford University Press
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Cloning and expression of cytosolic phosphoglycerate kinase from pea (Pisum sativum L.)
Received 25 July 2003; Accepted 16 December 2003
Department of Biological Sciences, University of Exeter, Exeter EX4 4PS, UK
* Present address: John Innes Centre for Plant Science Research, Colney Lane, Norwich NR4 7UH, UK. To whom correspondence should be addressed. Fax: +44 (0)1392 264668. J.A.Bryant{at}ex.ac.uk Present address: Department of Biochemistry, North Carolina State University, Raleigh, NC 27695, USA. 
Present address: Molecular Toxicology Laboratory, Imperial College School of Medicine, London SW7 2AZ, UK.
¶ Present address: Director, Henry Wellcome Centre for Biocatalysis, University of Exeter, Exeter EX4 4QD, UK.
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Abstract |
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In common with several other respiratory and photosynthetic enzymes, a sub-population of cytosolic phosphoglycerate kinase (PGK) occurs in the nucleus in pea leaves and shoots. The full-length cDNA encoding pea cytosolic PGK has been cloned and sequenced, revealing not only the PGK ‘signature’ but also a nuclear localization signal (NLS). A translational fusion of PGK and GFP was used to transform tobacco BY-2 cells resulting in GFP locating to the cell nuclei.
Key words: GFP, moonlighting proteins, nucleus, pea, phosphoglycerate kinase, Pisum sativum.
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There is increasing evidence from both prokaryotic and eukaryotic organisms that many proteins have more than one role (Jeffery, 1999). These bi-functional proteins have been termed ‘moonlighting’ proteins, although this does not necessarily imply that one role is more important than the other. It has been established in previous work that several respiratory and photosynthetic enzymes are present in the nuclei of pea leaves (Anderson et al., 1995). One of these enzymes is phosphoglycerate kinase (PGK) and in a recent detailed study using antibodies specific for the cytosolic and chloroplastic isozymes, both have been shown to occur in nuclei (Anderson et al., 2003; LE Anderson, JA Bryant, AA Carol, unpublished results). The role of PGK within pea nuclei has not been clearly established but there is preliminary evidence that it acts as an accessory protein to DNA polymerase-
(Bryant et al., 2000), a situation that has also been reported for certain mammalian cells (Jindal and Vishwanatha, 1990a, b; Popanda et al., 1998). As part of the investigation into the role of PGK in pea nuclei, the cDNA encoding the cytosolic PGK has been cloned, sequenced and expressed. The results of this work are presented here. A 525 bp fragment of the cytosolic PGK coding sequence was amplified by PCR from cDNA prepared from etiolated pea shoot apices. The primers were based on the consensus of other plant PGK sequences and the amplified fragment corresponded to amino acids 237–412 in the translated sequence (Fig. 1a). A full-length cDNA was obtained from this fragment using 3' and 5' RACE. The full-length cDNA was ligated into the Promega pGEM Easy vector at the multi-cloning site and the recombinant plasmid was amplified in E. coli. The sequence of the cDNA was determined by standard chain termination techniques. The sequence is lodged in the EMBL database (accession number AF275639 [GenBank] .1). The pea PGK sequence is 1501 bp long, containing a reading frame of 1203 bases and encoding a protein of 42 261 kDa. The PGK ‘signature’ (Watson and Littlechild, 1990) is clearly visible near the N-terminus (Fig. 1b). In relation to the dual location of the enzyme in pea, the sequence exhibits a very clear nuclear localization signal (Dingwall and Laskey, 1991) at positions 4–20 (Fig. 1c).
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In order to test whether the NLS was effective in vivo, the coding sequence was ligated to that of green fluorescent protein (GFP). The PGK coding sequence was on the 5' side of the GFP sequence and the TAA stop codon in PGK was changed to TAC in order to allow read-through. For expression in plant cells the hybrid gene was placed under the control of the CaMV 35S promoter in the pCAMBIA1302 plasmid vector. After amplification of the plasmid in E. coli, it was transferred to Agrobacterium tumefaciens by the direct freeze–thaw method of An (1986). The A. tumefaciens was then used to transform tobacco BY-2 cells as described by Shaul et al. (1996). GFP was visualized essentially as described by Neuhaus and Boevink (2001). In cells transformed with the hybrid gene, the nuclei were strongly fluorescent (Fig. 2); nuclei of cells transformed with GFP alone showed slight fluorescence and cells transformed with the ‘empty’ plasmid exhibited a very low background fluorescence (data not shown). These results show clearly that the NLS in PGK is able to transport the PGK-GFP hybrid protein into the nucleus. It is difficult to determine the relative total fluorescence of nucleus and cytosol, but in mammalian cells, biochemical analysis indicates that c. 10% of the PGK is in the nuclei (Vishwanatha et al., 1992). The strong nuclear fluorescence apparent in Fig. 2 may indicate a greater percentage than this, but it is possible that because of differences in protein folding, the NLS is more available in the hybrid protein than in the native protein. It thus remains unknown as to why a protein with a functional NLS is mostly, in nature, located in the cytosol. It is possible, that as with a small number of other proteins known to partition between cytoplasm and nucleus, phosphorylation at or near the NLS is important (Greenwood and Johnson, 1995; Tagawa et al., 1995). Thus, the prediction by Prosite (Bairoch, 1992) of possible target sites for protein kinase C (Woodgett et al., 1986) within the NLS at residues 3 and 9 is very interesting.
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Acknowledgements |
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We thank Dennis Francis and Hilary Rogers for the initial supply of tobacco BY-2 cells and for advice on their culture. The following are thanked for financial support via research grants for this work: BBSRC (DCB, JAB), and NATO (JAB, JAL). GD gratefully acknowledges scholarship funding from the Lithuanian Government, Fermentas (Vilnius), the UK ORS scheme, and the University of Exeter (Stratton Scholarship).
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