Regulation of peroxisomal metabolite carrier proteins

维持细胞的氧化还原状态对细胞的生存很重要，因为失衡会严重干扰细胞的几项功能。在之前的资助期间，我们在拟南芥中鉴定了一种过氧体载体，该载体在过氧酶体中提供与NAD的氧化还原反应。这种载体是控制过氧化体氧化还原动态平衡的一个很好的候选者。此外，我们还发现NAD载体经历了可逆的蛋白质磷酸化。这种翻译后修饰是对内部刺激和环境线索调整过氧化体NAD导入活性的快速反应。这里提出的项目将是理解过氧酶体氧化还原状态的调控网络的重要一步。我们解决了以下问题：(1)体内NAD载体的磷酸化对过氧化物体的功能有什么影响？具有过氧化体NAD供应缺陷的拟南芥和酵母突变体将被磷酸化或非磷酸化形式的目标蛋白补充。对由此产生的表型的分析将告诉我们，NAD载体的磷酸化状态在多大程度上影响生物体中过氧化物体NAD依赖的途径。(2)磷酸化和非磷酸化NAD载体蛋白的化学计量比定义了蛋白质活性变化的程度，从而影响了过氧化体功能？为了回答这个问题，我们将使用稳定的同位素进行基于质谱学的定量磷酸蛋白质组研究，并确定NAD载体的磷酸化事件。我们选择拟南芥幼苗作为研究对象，是因为在这些发育阶段需要高通量的NAD进入植物过氧体来动员储藏油。结果将为数学模型提供额外的信息，使我们能够描述细胞的调节网络。(3)哪种蛋白激酶负责通过磷酸化来调节过氧化体NAD载体？为了发现负责调节NAD载体活性的激酶，我们将进行光交联实验。这种方法将使我们能够有效地从拟南芥蛋白提取物中提取出激酶-底物复合体。相互作用的激酶将通过质谱学的蛋白质测序进行鉴定，并通过以NAD载体为底物的体外和体内激酶分析进行验证。这项拟议的工作将有助于我们实现将过氧化物体整合到细胞代谢中的长期目标。过氧体转运蛋白通过过氧体膜调节中间产物的流量，因此是重要的主开关，控制着过氧体和其他细胞室之间的溶质交换。然而，我们对这些承运人的监管知识有限。

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.