Biological roles of atpap2 in the mitochondria

Abstract

Arabidopsis thaliana Purple Acid Phosphatase 2 (AtPAP2) is anchored on the outer membranes of chloroplasts and mitochondria through its C-terminal transmembrane motif. Although the growth phenotypes of pap2 T-DNA line appear normal, the Arabidopsis thaliana overexpressing AtPAP2 line (OE7) bolts earlier and produces more seeds. To date, AtPAP2 is the only phosphatase that is known to be anchored on the outer membrane of the mitochondria. The objective of this study was to find out its biological roles in the mitochondria.

Cytosolic STY kinases phosphorylate the transit peptides of certain precursor proteins and regulate their import into chloroplasts. In this study, AtPAP2 was showed to interact with seven out of nine multiple organellar RNA editing factor (MORF) family proteins by yeast-two hybrid and bimolecular fluorescence complementation (BiFC) assays. The presequences of several precursor MORF (pMORF) family proteins, which play role in RNA editing in mitochondria and plastids, are phosphorylated by cytosolic STY kinases. STY-phosphorylated pMORF3, but not its non-phosphorylatable mutant (pMORF3-T17/20/35A), can complex with 14-3-3 and HSP70. The import rate of wheat germ lysate (WGL)-synthesized pMORF3-T17/20/35A into mitochondria was much faster than that of WGL-pMORF3 and the import rate of WGL-pMORF3 into mitochondria isolated from the pap2 T-DNA line was slower than that into mitochondria isolated from the wild-type plants. Therefore, STY kinases and AtPAP2 post-translationally regulate the import of the pMORF3 into the mitochondria.

Previous studies revealed that the photosynthetic rate of OE7 excess than wild type and its leaves contain higher amounts of ATP, malate, glutamine and asparagine than the wild type at the end of the night (T= 0) and the middle of the day (T= 8). In this study, the iTRAQ quantitative proteomics approach was used to investigate the impact of AtPAP2 overexpression on the mitochondrial proteome. A total of 903 mitochondrial proteins were identified from mitochondria isolated from 20-day-old leaves at T= 0 and T= 8. Proteins differentially expressed between the wild-type and OE7 mitochondria were identified in various pathways, including the respiratory electron chain, tricarboxylic acid (TCA) cycle, RNA editing and mitochondrial protein import components (MPIC). The lower protein abundances of complex I and V subunits at both time points and the lower in-gel activity of complex I at T= 8 in OE7 reflected that the efficiency of the ATP production in OE7 mitochondria may have decreased. At T= 8, OE7 mitochondria contained higher levels of metabolites (glutamine, asparagine and fumarate) and enzymes (mitochondrial isocitrate dehydrogenase 1 and mitochondrial malate dehydrogenase 1) required for nitrogen assimilation; whereas, at T= 0, the high ATP output from chloroplasts at daytime may have suppressed the TCA cycle in the OE line, as the abundances of TCA cycle enzymes between 2-oxoglutarate and malate/OAA in down-regulated when more ATP production is unnecessary.

In summary, these findings depict how AtPAP2 overexpression directly or indirectly affects the physiology of the mitochondria by regulating the import of precursor proteins into the mitochondria.