Purification and functional characterisation of COMATOSE a peroxisomal ABC transporter from Arabidopsis thaliana

高等生物如植物和动物的细胞被分成含有特定生物功能的区室。每个隔室都被一层膜包围，这层膜对大多数生物分子的运动起到了屏障的作用。为了使不同隔室的活动进行，必须发生分子跨膜的受控运输。在大多数情况下，转运是由膜内的蛋白质（转运蛋白）介导的，并且通常需要消耗能量来使转运的分子逆着浓度梯度移动。ABC转运蛋白是一大类膜蛋白，其利用由ATP分解释放的能量转运广泛的生物分子，ATP是活生物体的主要“能量货币”。这个建议是关于一个特定的ABC转运蛋白发现在膜的细胞室称为过氧化物酶体。过氧化物酶体具有一系列不同的生物学功能，但该项目特别感兴趣的是β-氧化。在这条途径中，脂肪样分子通过一次连续去除2个碳原子而被切碎。不同的分子可以出于不同的目的进行β-氧化，以提供能量，去除不需要的分子或产生具有不同生物活性的分子，如激素。植物、动物和酵母的过氧化物酶体膜含有类似的ABC转运蛋白。在酵母和植物中有相当好的证据表明，这些蛋白质参与将脂肪转运到过氧化物酶体中进行分解。在动物中也可能是这样，但证据并不那么清楚。有一点是清楚的，即有缺陷的过氧化物酶体ABC转运蛋白的植物和动物是非常病态的。出生时有缺陷的过氧化物酶体ABC转运蛋白（称为ALDP）的儿童可能会发展为严重的神经系统疾病，称为儿童脑X连锁肾上腺脑白质营养不良，导致进行性残疾和死亡，并且无法治愈和改善治疗有限。在相当于ABC转运蛋白（称为CTS）缺陷的植物种子不能发芽，除非使用特殊的技巧，有一些激素缺乏，比野生型植物繁殖力差。我们不知道植物、酵母和人类过氧化物酶体ABC转运蛋白在多大程度上做同样的事情，但由于它们可能有共同的进化起源，因此很有可能一些功能是保守的。我们将通过找出植物转运蛋白是否可以代替酵母转运蛋白发挥作用以及人类和酵母转运蛋白是否可以代替植物转运蛋白发挥作用来研究这一点。我们还将通过研究植物转运蛋白可以转运哪些分子以及蛋白质的哪些部分是这种活动所必需的来了解更多关于植物转运蛋白的精确功能。通过了解这种植物蛋白的活性，我们希望了解如何操纵它来生产改良的作物品种。了解植物，酵母和人类转运蛋白之间的相似性和差异也可能对人类转运蛋白的作用产生新的见解，这可能有助于了解X连锁肾上腺脑白质营养不良的病理学。

Cells of higher organisms such as plants and animals are divided into compartments that contain specific biological functions. Each compartment is surrounded by a membrane which acts as a barrier to movement of most biological molecules. In order for the activities of different compartments to proceed, the controlled transport of molecules across membranes has to occur. In most cases transport is mediated by proteins (transporters) within the membrane and often expenditure of energy is required to move the transported molecules against a concentration gradient. ABC transporters are a large class of membrane proteins which transport a wide range of biological molecules using energy released by breakdown of ATP, the principal 'energy currency' of living organisms. This proposal is concerned with one specific ABC transporter found in the membrane of the cellular compartment known as the peroxisome. Peroxisomes carry out a range of different biological functions but the one of particular interest to this project is beta-oxidation. In this pathway fat-like molecules are chopped up by sequential removal of 2 carbon atoms at a time. Different molecules may be subjected to beta-oxidation for different purposes - to provide energy, to remove unwanted molecules or to generate molecules with different biological activities, such as hormones. Peroxisome membranes of plants, animals and yeast contain similar ABC transporter proteins. There is quite good evidence in the case of yeast and plants that these proteins are involved in transporting fats into peroxisomes to be broken down. This may also be the case in animals, but the evidence is not so clear. What is clear is that plants and animals which have defective peroxisomal ABC transporters are very sick. Children born with a defective peroxisome ABC transporter called ALDP may develop a severe neurological disorder called Childhood Cerebral X-linked Adrenoleukodystrophy which results in progressive disability and death, and for which there is no cure and limited ameliorative treatment. Plant seeds with a defect in the equivalent ABC transporter (called CTS) can't germinate unless special tricks are used, have some hormone deficiencies and are less fertile than wild-type plants. We don't know to what extent the plant, yeast and human peroxisomal ABC transporters do the same things, but since they probably share a common evolutionary origin there is a good chance some functions are conserved. We will seek to investigate this by finding out if the plant transporter can function in place of the yeast transporter and if the human and yeast transporters can function in place of the plant transporter. We will also find out more about the precise functions of the plant transporter by investigating which molecules it can transport and what parts of the protein are necessary for this activity. By learning about the activity of this plant protein we would hope to understand how we could manipulate it to produce improved crop varieties. Understanding the similarities and differences between the plant, yeast and human transporters might also yield new insights into what the human transporter is doing which could be of benefit to understanding the pathology of X-linked Adrenoleukodystrophy.