The role of a peroxisomal NAD+ carrier in plants

NAD+ is an essential cofactor for several metabolic processes. In plants, the NAD+ biosynthesis takes exclusively place in the cytosol (Noctor et al., 2006; Hashida et al., 2009) whereas NAD+-dependent enzymes are located in the cytosol and other cell compartments, including peroxisomes. Earlier evidences assume that the peroxisomal membrane is impermeable for “bulky” cofactors like NAD+ and CoA (Donaldson, 1981; Antonenkov and Hiltunen, 2012). However, the fact that peroxisomal metabolism require theses molecules (e.g. for fatty acid β-oxidation, photorespiration, and H2O2 detoxification; Hu et al., 2012) necessitates the need for a NAD+ import. For decades, the existence of such a peroxisomal NAD+ transport protein was controversially discussed. In this thesis, the first peroxisomal NAD+ carrier, named PXN, was characterized in the model plant Arabidopsis thaliana. The carrier encoded by the gene At2g39970 is a member of the mitochondrial carrier family (MCF) and closely related in sequence to peroxisomal, chloroplastic, and mitochondrial ATP and NAD+ transporters from Arabidopsis, yeast, and human. Interaction studies confirmed that PXN is targeted to peroxisomes via interaction with PEX19 that is known to act as chaperone for PMP targeting (Jones et al., 2004; Van Ael and Fransen, 2006). Performing uptake assays, it was demonstrated that recombinant PXN protein catalyzed the transport of NAD+ and CoA in exchange with AMP, which was recently confirmed by Agrimi et al. (2012b). Furthermore, cell-free expressed PXN protein mediated the exchange of NAD+ with NADH and acts thereby as reduction/oxidation (redox) shuttle. The NADH transport activity is unique compared to the other known MCF-type NAD+ transporter from Arabidopsis, yeast, and human. This thesis indicated first evidences that the PXN activity is positively regulated via phosphorylation. The phosphorylation site at serine 155 was detected in two independent phosphoproteomic studies (Eubel et al., 2008; Ito et al., 2009). Here, it was in addition shown that the phosphorylation site at S155 and the unique loop region of the PXN protein did not influence the PEX19-dependent peroxisomal targeting, indicating that the PEX19 binding site is not located at the loop region. Three independent Arabidopsis pxn T-DNA insertion lines illustrated the requirement of peroxisomal NAD+ and CoA supply mediated by PXN for optimal fatty acid β-oxidation. pxn seedlings were delayed in breakdown of fatty acids released from storage oil, which was visualized by higher levels of storage fatty acids and the retention of oil bodies. However, germination was not impaired in plants deficient in PXN. The pxn β-oxidation phenotype correlated with the increased PXN expression during β-oxidation-dependent seedling establishment and the PXN co-expression with proteins involved in β-oxidation. Furthermore, the link between PXN function and the β-oxidation-dependent processes (e.g. mobilization of storage oil and biosynthesis of phytohormones) was exhibited by the expression pattern using PXN promoter-GUS studies in stable transformed Arabidopsis seedlings. A redundant system for PXN was predicted, because pxn mutants exhibited an impaired but not complete block of β-oxidation, as expected for plants lacking the NAD+ and CoA supply via PXN. Two redundant systems were assumed. (i) The NAD+/AMP shuttle could be taken over by the peroxisomal adenine nucleotide carriers PNC1 and PNC2 when PXN is absent, because both transport substrates, ATP and NAD+, are similar in structure. To address this, mutant lines repressing both pnc genes and pxn were generated by using a small interfering RNA approach. The obtained transgenic plants needs to be investigated in the future. (ii) The NAD+/NADH exchange function of PXN could be redundant to the malate/oxaloacetate (malate/OAA) shuttle that transfers reducing equivalents across the peroxisomal membrane (Pracharoenwattana et al., 2007). Furthermore, the peroxisomal hydroxypyruvate reductase 1 (HPR1) is proposed to play a role in regeneration NAD+ for β-oxidation and thus also functions as redox shuttle during early seedling growth (Pracharoenwattana et al., 2010). To test this hypothesis, different Arabidopsis plants were generated in this work by crossing the pxn lines with the mutant defective in both pMDHs and HPR1 and by repressing the PXN expression using an PXN specific artificial microRNA in the pmdh and hpr1 mutant lines. However, the resulted transgenic plants need to be further investigated regarding the impact on β-oxidation for storage oil mobilization. In silico analysis revealed that PXN is co-expressed with the ascorbate peroxidase (APX) and the NADH-dependent monodehydroascorbate reductase (MDAR), which are required for H2O2 detoxification (Graham, 2008). PXN expression is altered during oxidative stress, when H2O2 accumulated inside the cells. This implicated a role of PXN in supplying NAD+ for peroxisomal H2O2 detoxification via the APX/MDAR system. This hypothesis has to be verified by examining pxn mutants under oxidative stress conditions. In sum, this thesis described the characterization and the function of a peroxisomal NAD+ and CoA carrier during seedling establishment and oxidative stress. Site-directed mutagenesis of the PXN protein revealed that the transport activity is regulated by phosphorylation. Regarding the PMP import, it was demonstrated that the PEX19 receptor is involved in the peroxisomal targeting of PXN and several other PMPs.