Summary: The existence of a transport protein that imports cytosolic NAD⁺ into peroxisomes has been controversially discussed for decades. Nevertheless, the biosynthesis of NAD⁺ in the cytosol necessitates the import of NAD⁺ into peroxisomes for numerous reduction/oxidation (redox) reactions. However, a gene encoding such a transport system has not yet been identified in any eukaryotic organism. Here, we describe the peroxisomal NAD⁺ carrier in Arabidopsis. Our candidate gene At2g39970 encodes for a member of the mitochondrial carrier family. We confirmed its peroxisomal localization using fluorescence microscopy. For a long time At2g39970 was assumed to represent the peroxisomal ATP transporter. In this study, we could show that the recombinant protein mediated the transport of NAD⁺. Hence, At2g39970 was named PXN for peroxisomal NAD⁺ carrier. The loss of PXN in Arabidopsis causes defects in NAD⁺‐dependent β‐oxidation during seedling establishment. The breakdown of fatty acid released from storage oil was delayed, which led to the retention of oil bodies in pxn mutant seedlings. Based on our results, we propose that PXN delivers NAD⁺ for optimal fatty acid degradation during storage oil mobilization.

Plant peroxisomes are unique subcellular organelles which play an indispensable role in several key metabolic pathways, including fatty acid β‐oxidation, photorespiration, and degradation of reactive oxygen species. The compartmentalization of metabolic pathways into peroxisomes is a strategy for organizing the metabolic network and improving pathway efficiency. An important prerequisite, however, is the exchange of metabolites between peroxisomes and other cell compartments. Since the first studies in the 1970s scientists contributed to understanding how solutes enter or leave this organelle. This review gives an overview about our current knowledge of the solute permeability of peroxisomal membranes described in plants, yeast, mammals and other eukaryotes. In general, peroxisomes contain in their bilayer membrane specific transporters for hydrophobic fatty acids (ABC transporter) and large cofactor molecules (carrier for ATP, NAD and CoA). Smaller solutes with molecular masses below 300–400 Da, like the organic acids malate, oxaloacetate, and 2‐oxoglutarate, are shuttled via non‐selective channels across the peroxisomal membrane. In comparison to yeast, human, mammals and other eukaryotes, the function of these known peroxisomal transporters and channels in plants are discussed in this review.

Cofactors such as NAD, AMP, and Coenzyme A (CoA) are essential for a diverse set of reactions and pathways in the cell. Specific carrier proteins are required to distribute these cofactors to different cell compartments, including peroxisomes. We previously identified a peroxisomal transport protein in Arabidopsis (Arabidopsis thaliana) called the peroxisomal NAD carrier (PXN). When assayed in vitro, this carrier exhibits versatile transport functions, e.g. catalyzing the import of NAD or CoA, the exchange of NAD/NADH, and the export of CoA. These observations raise the question about the physiological function of PXN in plants. Here, we used Saccharomyces cerevisiae to address this question. First, we confirmed that PXN, when expressed in yeast, is active and targeted to yeast peroxisomes. Secondl, detailed uptake analyses revealed that the CoA transport function of PXN can be excluded under physiological conditions due to its low affinity for this substrate. Third, we expressed PXN in diverse mutant yeast strains and investigated the suppression of the mutant phenotypes. These studies provided strong evidences that PXN was not able to function as a CoA transporter or a redox shuttle by mediating a NAD/NADH exchange, but instead catalyzed the import of NAD into peroxisomes against AMP in intact yeast cells.

The metabolite transporter GPT1 is important for NADPH provision in both plastid and peroxisomes, mostly during pollen tube growth towards ovules, and is thus e

In land plants, peroxisomes play key roles in various metabolic pathways, including the most prominent examples, that is lipid mobilization and photorespiration. Given the large number of substrates that are exchanged across the peroxisomal membrane, a wide spectrum of metabolite and cofactor transporters is required and needs to be efficiently coordinated. These peroxisomal transport proteins are a prerequisite for metabolic reactions inside plant peroxisomes. The entire peroxisomal “permeome” is closely linked to the adaption of photosynthetic organisms during land plant evolution to fulfill and optimize their new metabolic demands in cells, tissues, and organs. This review assesses for the first time the distribution of these peroxisomal transporters within the algal and plant species underlining their evolutionary relevance. Despite the importance of peroxisomal transporters, the majority of these proteins, however, are still unknown at the molecular level in plants as well as in other eukaryotic organisms. Four transport proteins have been recently identified and functionally characterized in Arabidopsis so far: one transporter for the import of fatty acids and three carrier proteins for the uptake of the cofactors ATP and NAD into plant peroxisomes. The transport of the three substrates across the peroxisomal membrane is essential for the degradation of fatty acids and fatty acids-related compounds via β-oxidation. This metabolic pathway plays multiple functions for growth and development in plants that have been crucial in land plant evolution. In this review, we describe the current state of their physiological roles in Arabidopsis and discuss novel features in their putative transport mechanisms.