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The essential micronutrient manganese (Mn) functions as redox-active cofactor in active sites of enzymes and, thus, is involved in various physiological reactions. Moreover, in oxygenic photosynthetic organisms, Mn is of special importance, since it is central to the oxygen-evolving complex in photosystem II. Although Mn is an essential micronutrient, increased amounts are detrimental to the organism; thus, only a small window exists for beneficial concentrations. Accordingly, Mn homeostasis must be carefully maintained. In contrast to the well-studied uptake mechanisms in cyanobacteria, it is largely unknown how Mn is distributed to the different compartments inside the cell. We identified a protein with so far unknown function as a hypothetical Mn transporter in the cyanobacterial model strain Synechocystis sp. PCC 6803 and named this protein Mnx for Mn exporter. The knockout mutant Δmnx showed increased sensitivity toward externally supplied Mn and Mn toxicity symptoms, which could be linked to intracellular Mn accumulation. 54Mn chase experiments demonstrated that the mutant was not able to release Mn from the internal pool. Microscopic analysis of a Mnx::yellow fluorescent protein fusion showed that the protein resides in the thylakoid membrane. Heterologous expression of mnx suppressed the Mn-sensitive phenotype of the Saccharomyces cerevisiae mutant Δpmr1. Our results indicate that Mnx functions as a thylakoid Mn transporter and is a key player in maintaining Mn homeostasis in Synechocystis sp. PCC 6803. We propose that Mn export from the cytoplasm into the thylakoid lumen is crucial to prevent toxic cytoplasmic Mn accumulation and to ensure Mn provision to photosystem II.

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 essential micronutrient manganese (Mn) functions as redox-active cofactor in active sites of enzymes and, thus, is involved in various physiological reactions. Moreover, in oxygenic photosynthetic organisms, Mn is of special importance, since it is central to the oxygen-evolving complex in photosystem II. Although Mn is an essential micronutrient, increased amounts are detrimental to the organism; thus, only a small window exists for beneficial concentrations. Accordingly, Mn homeostasis must be carefully maintained. In contrast to the well-studied uptake mechanisms in cyanobacteria, it is largely unknown how Mn is distributed to the different compartments inside the cell. We identified a protein with so far unknown function as a hypothetical Mn transporter in the cyanobacterial model strain