Reaserch Of Protein Interaction Of A Complex With Alternative NAD(P)H Dehydrogenase Activity From Arabidopsis Thaliana Mitochondria

Mitochondria are important energy production unit of cells, which are the basic unit of life. Mitochondria result in formation of ATP through oxidative phosphorylation. Plant mitochondria contain multiple energy dissipating pathways, including alternative oxidase pathway, uncoupling pathway and alternative NAD(P)H dehydrogenase pathway. Alternative NAD(P)H dehydrogenases are similar to mitochondria electron transport chain complex I in transferring electron to oxygen along electron transport chain. However, these alternative NAD(P)H dehydrogenases do not directly contribute to proton pumping and ATP synthesis, and their activities cannot be inhibited by rotenone, the specific inhibitor of complex I, so they are also called rotenone-insensitive NAD(P)H dehydrogenase. In recent years, much progress have been made for separating alternative NAD(P)H dehydrogenase. Recently, proteins are coming back into focus. Studies of proteins on protein complex level can not only reveal functions to unknown proteins, but also provide opportunity to investigate novel functions for known proteins in different protein complexes.The present study takes model plant Arabidopsis thaliana as study object and separates one protein complex from soluble fraction of Arabidopsis thaliana mitochondria by using native-PAGE. This complex possesses a activity which is similar to alternative NAD(P)H dehydrogenase. We have identified the components of this protein complex with MALDI-TOF and found that besides lipoamide dehydrogenase 2 (LPD2). there are two other subunit, N-acetyl-γ-glutamyl-phosphate reductase (NAGPR) and molecular chaperonin Cpn20, in this protein complex. We then detect the interactions between LPD2, Cpn20 and NAGPR by co-immunoprecipitate (co-IP). Both LPD2 and Cpn20 can be co-purified when using NAGPR antibody, while only Cpn20 or LPD2 can be purified when using LPD2 or Cpn20 antibody. These results suggest the interaction between LPD2 and Cpn20 is direct and the interactions containing NAGPR need study one step further. We take advantage of tandem affinity purification to investigate the interaction between LPD2. Cpn20 and NAGPR in vivo. In a transient expression system of Nicotianabenthamiana, the perfect expression levels of LPD2-TAPa and NAGPR-TAPa fusionproteins imply that no mutation occurs during expression vector construction and thetarget proteins can express successfully with a TAP tag. However, in this transientexpression system, the protein complexes purified by tandem affinity purification allshow a significant non-specific band, which suggest that transient expression systemof N. benthamiana is unsuitable to study exogenous protein complexes and proteininteractions. Thereby we carry out tandem affinity purification in stable expressionsystem of A. thaliana to identify the protein interaction in vivo. The proteincomplexes containing LPD2-TAPa and NAGPR-TAPa respectively are purified byIgG Beads, and separated by SDS-PAGE, then detected by western-blot. The resultsverify the specific interaction between LPD2 and Cpn20, and identify NAGPRcombines with LPD2-Cpn20 complex non-specifically.These results suggest that in the complex which is separated from soluble fractionof A. thaliana mitochondria, Cpn20 can directly interact with LPD2. This is the firsttime to reveal the interaction between them. Cpn20 may help LPD2 folding after ittransport in mitochondria. The interaction between them probably relates to thesignificant function of LPD2, such as thermogenesis, stress resistance, avoidance ofreactive oxygen species formation and regulation of the reduction state when it act asalternative NAD(P)H dehydrogenase in plant, or is involved in response of plant tostress.