First published online February 6, 2003; 10.1104/pp.102.015081
Plant Physiol, March 2003, Vol. 131, pp. 1033-1041
Dual Intracellular Localization and Targeting of Aminoimidazole
Ribonucleotide Synthetase in Cowpea1
Danica Erin
Goggin,
Richard
Lipscombe,
Elena
Fedorova,
A. Harvey
Millar,
Anthea
Mann,
Craig Anthony
Atkins,* and
Penelope Mary Collina
Smith
Department of Botany (D.E.G., E.F., A.M., C.A.A., P.M.C.S.) and
Biochemistry and Molecular Biology (A.H.M.), The University of
Western Australia, 35 Stirling Highway, Crawley, Western Australia
6009, Australia; and Proteomics International Pty Ltd, Level 21, 197 St
Georges Terrace, Perth, Western Australia 6000, Australia
(R.L.)
De novo purine biosynthesis is localized to both mitochondria and
plastids isolated from Bradyrhizobium sp.-infected cells of cowpea (Vigna unguiculata L. Walp) nodules, but
several of the pathway enzymes, including aminoimidazole ribonucleotide
synthetase (AIRS [EC 6.3.3.1], encoded by Vupur5), are
encoded by single genes. Immunolocalization confirmed the presence of
AIRS protein in both organelles. Enzymatically active AIRS was purified
separately from nodule mitochondria and plastids. N-terminal sequencing
showed that these two isoforms matched the Vupur5 cDNA
sequence but were processed at different sites following import; the
mitochondrial isoform was five amino acids longer than the plastid
isoform. Electrospray tandem mass spectrometry of a trypsin digest of
mitochondrial AIRS identified two internal peptides identical with the
amino acid sequence deduced from Vupur5 cDNA. Western
blots of proteins from mitochondria and plastids isolated from root
tips showed a single AIRS protein present at low levels in both
organelles. 35S-AIRS protein translated from a
Vupur5 cDNA was imported into isolated pea (Pisum
sativum) leaf chloroplasts in vitro by an ATP-dependent process
but not into import-competent mitochondria from several plant and
non-plant sources. Components of the mature protein are likely to be
important for import because the N-terminal targeting sequence was
unable to target green fluorescent protein to either chloroplasts or
mitochondria in Arabidopsis leaves. The data confirm localization of
the protein translated from the AIRS gene in cowpea to both plastids
and mitochondria and that it is cotargeted to both organelles, but the
mechanism underlying import into mitochondria has features that are yet
to be identified.
1
This work was supported by the Australian
Research Council (grants to C.A.A. and P.M.C.S.) and by an Australian
Postgraduate Award (to D.E.G.).
*
Corresponding author; e-mail catkins{at}cyllene.uwa.edu.au; fax
61-8-93801001.
© 2003 American Society of Plant Biologists
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