Supplying plant peroxisomes with energy

ATP代表细胞的主要能量货币，驱动大量的代谢反应。它的可用性和持续供应对细胞代谢有着至关重要的影响。由于ATP的合成仅限于植物中的少数细胞区室，因此ATP必须被运输进出不能合成它的细胞区室。特定的载体蛋白介导细胞内ATP交换，因此对多种代谢途径至关重要。在我们的前期研究中，我们对拟南芥中两种能够将ATP导入过氧化物酶体的ATP转运蛋白进行了研究，在本研究中，我们将解决以下主要问题以了解过氧化物酶体ATP导入的功能：（1）哪些ATP消耗反应依赖于过氧化物酶体ATP？我们证明，拟南芥植物过氧化物酶体ATP进口受损无法代谢他们的存储油。结果，突变体幼苗的生长被阻止。我们的初步研究结果还表明，过氧化物酶体ATP是至关重要的有毒代谢产物丙酸的降解。我们将进行13C标记实验和有针对性的代谢物分析，以阐明过氧化物酶体参与这一研究较少的途径。(2)ATP进入过氧化物酶体的驱动力是什么？我们证明了ATP与ADP或AMP交换转运。这样的反交换将产生穿过过氧化物酶体膜的膜电位，其抵消ATP向过氧化物酶体的连续供应。我们推测，ATP进口过程中的负电荷的内向净转移是由质子运输平衡。我们将在本提案中检验这一假设。(3)ATP的供给和需求如何影响过氧化物酶体代谢？我们将评估ATP池在拟南芥过氧化物酶体在体内使用基于GFP的ATP传感器在不同的突变体植物。此外，我们将在过氧化物酶体基质中加入ATP水解酶，以加强ATP吸收。这将使我们能够深入了解植物过氧化物酶体中ATP库的动态变化，从而进一步阐明ATP对过氧化物酶体代谢的影响。

ATP represents the major energy currency of the cell, driving a large number of metabolic reactions. Its availability and continuous provision has a crucial impact on cellular metabolism. Since ATP synthesis is restricted to only a few cell compartments in plants, ATP has to be transported in and out of cell compartments that are unable to synthesize it. Specific carrier proteins mediate the intracellular ATP exchange and thus are crucial for multiple metabolic pathways. In our previous funding period we characterized two ATP transport proteins in Arabidopsis that are able to import ATP into peroxisomes.In this proposal we will address the following major questions to understand the function of peroxisomal ATP import: (1) Which ATP-consuming reactions depend on peroxisomal ATP? We demonstrated that Arabidopsis plants with an impaired peroxisomal ATP import were unable to metabolize their storage oil. As a consequence the mutant seedlings were arrested in their growth. Our preliminary results also indicate that peroxisomal ATP is crucial for the degradation of the toxic metabolite propionic acid. We will perform 13C-labelling experiments and targeted metabolite analyses to elucidate the involvement of peroxisomes in this less-studied pathway. (2) What is the driving force for ATP import into peroxisomes? We demonstrated that ATP is transported in exchange with ADP or AMP. Such a counter-exchange would generate a membrane potential across the peroxisomal membrane that counteracts the continuous supply of ATP into peroxisomes. We hypothesize that the inward-directed net-transfer of negative charges during ATP import is balanced by proton transport. We will test this hypothesis in this proposal. (3) How does supply and demand of ATP impact peroxisomal metabolism? We will assess the ATP pool in Arabidopsis peroxisomes in vivo by using a GFP-based ATP sensor in diverse mutant plants. In addition, we will implement an ATP-hydrolyzing enzyme in the peroxisomal matrix to intensify the ATP sink. This will allow us to get insights in dynamic changes of the ATP pool in plant peroxisomes, which will further elucidate the impact of ATP on peroxisomal metabolism.