Regulation of peroxisomal metabolite carrier proteins

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Maintaining the cellular redox status is important for cell survival, because an imbalance severely disturbs several cellular functions. In the previous funding period, we characterized a peroxisomal carrier in Arabidopsis that supplies redox reactions with NAD in peroxisomes. This carrier represents a good candidate for controlling the peroxisomal redox homeostasis. Furthermore, we showed that the NAD carrier undergoes reversible protein phosphorylation. Such a post-translational modification is a fast response to adjust the peroxisomal NAD import acativies to internal stimuli and environmental cues. The project proposed here will be a major step in understanding the regulatory network of the peroxisomal redox status. We address the following questions: (1) What are the consequences of the phosphorylation of the NAD carrier for the function of peroxisomes in vivo? Arabidopsis and yeast mutant with a defect in peroxisomal NAD supply will be complemented with the target protein in the phosphorylated or non-phosphorylated form. Analyses of the resulting phenotypes will tell us to what extent the phosphorylation status of the NAD carrier impacts peroxisomal NAD-dependent pathways in living organisms. (2) Which stoichiometric ratio of phosphorylated and non-phosphorylated NAD carrier protein defines the extent of changes in protein activity and thus effecting peroxisomal functions? To answer this, we will perform mass spectrometry-based quantitative phospho-proteome studies using stable isotopes and determine the phosphorylation events of the NAD carrier. We choose Arabidopsis seedlings as research objectives, because a high flux of NAD into plant peroxisomes is required for storage oil mobilization at these developmental stages. The results will provide additional information for a mathematical model that allows us to describe the regulatory network of the cell. (3) Which protein kinase is responsible for regulating the peroxisomal NAD carrier by phosphorylation? To discover the kinase responsible for regulating the activity of the NAD carrier, we will conduct photo-cross-linking experiments. This approach will allow us to efficiently pull-down the kinase-substrate complex from Arabidopsis protein extracts. The interacting kinase will be identified by protein sequencing via mass spectrometry and verified by in vitro and in vivo kinase assays using NAD carrier as a substrate.This proposed work will contribute to our long-term goal towards the integration of peroxisomes in the cellular metabolism. Peroxisomal transport proteins mediate the flux of intermediates across the peroxisomal membrane and thus are important master switches, controlling the exchange of solutes between peroxisomes and other cellular compartments. However, our knowledge about the regulation of these carrier is limited.