1 Heat Stress Response in Pea Involves Interaction of Mitochondrial Nucleoside Diphosphate Kinase with a Novel 86-Kilodalton Protein

2 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

3  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). 1. INTRODUCTION

4  NDPK isoforms have been found in the matrix as well as in the inter-membrane 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).

5  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.

6 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 Håkansson, 1999); lane 6, 25µg of purified chloroplasts. 2. Tissue-Specific Expression of the Pea mtNDPK

7 Figure 2. Immunolocalization of the pea mtNDPK in flower bud and young pea leaf. Positive fluorescent immunolabelling of the mtNDPK is seen as bright green spots. 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, petals; SE, sepals; UM, upper mesophyll; LM, lower mesophyll; V, vein. Scale bars = 100 µ m.

8 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] H 2 O 2 ); lane 4, heat stress (42°C); lane 4, cold stress (4°C). 3. An 86-kD Protein Is Newly Synthesized upon Heat Stress and Coprecipitates with the Pea mtNDPK

9 Figure 4. Analysis of de novo synthesized protein in pea upon heat and cold stress. A, PhosphorImage of immunoprecipitation of [ 35 S]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 [ 35 S]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] H 2 O 2 ); lane 4, heat stress (42°C); lane 4, cold stress (4°C).

10 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.