Heat Stress Response in Pea Involves Interaction
of Mitochondrial Nucleoside Diphosphate Kinase
with a Novel 86-Kilodalton Protein
1
Outline
1. INTRODUCTION
2. Tissue-Specific Expression of the Pea mtNDPK
3. An 86-kD Protein Is Newly Synthesized upon Heat Stress and
Coprecipitates with the Pea mtNDPK
4. The sequencing of the 86-kD protein
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1. INTRODUCTION

Nucleoside diphosphate kinases (NDPKs) are ubiquitous enzymes that
transfer phosphate groups from triphosphate nucleosides to nucleoside
diphosphates (NDPs) (Parks and Agarwal, 1973).

Recent reports have revealed the involvement of animal NDPKs in other
vital processes such as control of cell proliferation (Cipollini et al., 1997),
regulation of transcription (Postel et al., 1993; Ji et al., 1995), and protein
phosphotransferase (Engel et al., 1998; Wagner and Vu, 2000).

Interactions of NDPKs with different types of proteins have been reported
in several cases(Engel et al., 1998 ; Choi et al., 1999 ; Leung and
Hightower, 1997 ; Barthel and Walker, 1999).
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
NDPK isoforms have been found in the matrix as well as in the intermembrane space of mitochondria (Troll et al., 1993; Lambeth et al., 1997;
Milon et al., 1997; Struglics and Hakansson, 1999).

In animals, matrix NDPK isoforms have been suggested to catalyze transfer
of the phosphoryl group from GTP, produced by the TCA cycle, to ATP
(Herbert et al., 1955).

Struglics and Hakansson (1999) purified the first plant mitochondrial NDPK
isoform and suggested an inter-membrane space localization. This 17-kD
isoform, purified from pea (Pisum sativum L. cv Oregon sugarpod)
mitochondria, shows auto-phosphorylation on Ser residues, which is
characteristic of the human NDPK isoforms involved in signal transduction
(McDonald et al., 1993; Postel et al., 1993).
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
Expression of NDPKs varies between different tissues and developmental
stages.

Studies on heat-stress response in plant mitochondria have been mainly
focused on characterization of heat shock proteins (HSPs) (Neumann et al.,
1993; Lund et al., 1998). Only one mitochondrial small heat shock
protein(Downs and Heckathorn, 1998) has been functionally studied.

Investigations of the possible involvement of other proteins such as NDPK
in mitochondrial response to heat stress are therefore relevant.

The purpose of this work was to functionally characterize pea mtNDPK,
investigating tissue specificity in expression as well as a possible role in
response to different kinds of stress.

We also report a novel interaction of a 86-kD protein with the pea mtNDPK
under heat stress, providing evidence for a role of this mitochondrial NDPK
isoform in stress response in plants.
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2. Tissue-Specific Expression of the Pea mtNDPK
Figure 1. Western analysis of the pea mtNDPK in various subcellular fractions.
Lane 1, 25µg of flower mitochondria; lane 2, 25µg of root mitochondria;
lane 3, 25 µg of 7-d-old leaves mitochondria; lane 4, 25µg of 9-d-old
leaves mitochondria; lane 5, purified pea mtNDPK (according to Struglics
and Håk ansso n, 1 9 9 9); la ne 6 , 2 5µ g o f p u rifi ed chlo ro p last s .
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Figure 2. Immunolocalization of the
pea mtNDPK in flower bud and
young pea leaf. Positive fluorescent
immunolabelling of the mtNDPK is
seen as bright gr een spot s. A
through D, Flower bud; E and F,
nonexpanded pea leaf.
The pictures represent the following:
A, longitudinal flower bud section
incubated with anti-mtNDPK; B,
longitudinal flower bud section
incubated with preimmune serum; C,
transversal anthers section
incubated with anti-mtNDPK; D,
transversal anthers section
incubated with preimmune serum; E,
transversal young pea leaf section
incubated with anti-mtNDPK; and F,
transversal young pea leaf section
incubated with preimmune serum. A,
Anthers; O, ovary; ST, stamen; P,
p et als ; SE, sep als ; UM, upp er
mesophyll; LM, lower mesophyll; V,
vein. Scale bars = 100 µm.
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3. An 86-kD Protein Is Newly Synthesized upon Heat Stress and
Coprecipitates with the Pea mtNDPK
Figure 3. Western analysis of the pea mtNDPK in crude mitochondria prepared
from pea leaves exposed to various stress conditions for 4 h. Lane 1, control;
lane 2, high salt stress (400 mM NaCl); lane 3, oxidative stress (2% [v/v] H2O2);
lane 4, heat stress (42°C); lane 4, cold stress (4°C).
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Figure 4. Analysis of de novo synthesized protein in pea upon heat and
cold stress. A, PhosphorImage of immunoprecipitation of [35S]Met-labeled
crude mitochondrial proteins using the pea mtNDPK antibody. Lane
1, Control; lane 2, heat stress (42°C); lane 3, cold stress (4°C). B,
PhosphorImage of the time course of incorporation of [35S]Met into the
86-kD heat stress up-regulated protein, immunoprecipitated using the pea
mtNDPK antibody. Lane 1, 2 h; lane 2, 4 h; lane 3, 8 h. C, Western blot of
immunoprecipitations of crude mitochondrial proteins prepared from pea
leaves exposed to various stresses probed with anti-mtNDPK. Lane
1, Control; lane 2, high salt stress (400 mM NaCl); lane 3, oxidative stress
(2% [v/v] H2O2); lane 4, heat stress (42°C); lane 4, cold stress (4°C).
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4. The sequencing of the 86-kD protein
 Using mass spectrometry, sequences of trypsin digested peptides
were obtained.
 The sequences obtained were TWFM(L/I), ATGTVT(L/I) V, and (L/I)
SVPTS(L/I).
 Leu and Ile have the same molecular mass and can hence not be
distinguished using this method.
 However, analysis of the sequences revealed no similarity with other
proteins found in the databases, making identification of the 86-kD
heat up-regulated protein impossible.

We conclude that the 86-kD protein is an as yet uncharacterized
novel protein.
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