The Role of Protein-Protein Interactions in Sucrose Transport

植物有机体的一个重要特征是光合作用产生蔗糖。从本质上说，大气中的碳被同化成蔗糖，然后被输送到整个工厂，在那里它被用作能源。植物的生产力或产量直接取决于碳在蔗糖中的净吸收，以及最终有多少蔗糖被运输到植物的可收获部分，如种子。了解蔗糖是如何通过植物运输的，以及这种运输是如何调控的，对于了解基础植物生物学和优化植物生产力都是至关重要的。大豆子叶中的蔗糖运输蛋白似乎与植物细胞胞浆中的另一种蛋白相互作用。这种相互作用的蛋白质包含至少三个锚蛋白重复基序，它们通常参与将膜运输蛋白连接到细胞细胞骨架。由于蔗糖转运蛋白与其他蛋白质相互作用，这种相互作用可能在蔗糖转运蛋白在细胞表面的组装或在输入蔗糖并进一步代谢蔗糖的蛋白质复合体的形成/分解中起重要作用。因此，蔗糖运输可能在一定程度上受到转运蛋白和这个相互作用伙伴之间相互作用的性质的调节。本研究试图阐明这种蛋白质-蛋白质相互作用在蔗糖运输中的功能意义。三个主要目标如下：目标1.鉴定与锚蛋白样蛋白相互作用的蛋白质。为了了解这种相互作用的本质，重要的是要确定其他蛋白质是否也可能与植物蔗糖转运蛋白接触。例如，如果蔗糖代谢酶是蔗糖转运蛋白复合体的一部分，那么蔗糖输入可能直接与其随后的代谢有关。这些实验将解决这种可能性。目标2。评估蛋白质-蛋白质相互作用在蔗糖吸收中的作用。如果蔗糖转运蛋白与锚蛋白样蛋白的相互作用对植物中蔗糖的吸收有影响，那么锚蛋白样蛋白功能的中断应该以不同的方式干扰蔗糖运输。利用基因沉默技术和蔗糖摄取参数的测定，将破坏锚蛋白样蛋白的功能。目的#3.研究蔗糖转运蛋白和锚蛋白样蛋白的亚细胞定位。通过描述蔗糖转运蛋白的性质：锚蛋白样蛋白(ST：ALP)在细胞中的相互作用，解决了几个重要的问题。ST：ALP复合体在子叶发育过程中会发生变化吗？ST：ALP复合体是在什么时候形成的，与蔗糖转运蛋白和Ankryin样蛋白的合成有关？我们的mRNA分析表明，在子叶发育过程中，类锚定蛋白比蔗糖转运蛋白积累得更早，这就提出了一个问题，即蔗糖转运蛋白在翻译时是否与锚蛋白样蛋白结合。对蔗糖转运蛋白、锚蛋白样蛋白和其他潜在相互作用蛋白的免疫细胞化学分析，共聚焦和电子显微镜将解决这些问题。

One defining feature of plant organisms is the photosynthetic production of sucrose. Essentially, carbon from the atmosphere is assimilated into sucrose which is then transported throughout the plant where it is used as an energy source. The productivity, or yield, of plants directly depends on the net assimilation of carbon into sucrose and ultimately how much of this sucrose is transported to the harvestable components of plants, such as the seed. Understanding how sucrose is transported through the plant and how this transport is regulated is critical for both for understanding fundamental plant biology and optimization of plant productivity.The sucrose transport protein from soybean cotyledons appears to interact with another protein in the cytosol of plant cells. This interacting protein contains at least three ankyrin repeat motifs which often are involved in linking membrane transport proteins to the cellular cytoskeleton. Since the sucrose transporter interacts with other proteins, it may be that this interaction is important in the assembly of sucrose transporters on the cell surface or in the formation/disassembly of protein complexes that both import sucrose and metabolize it further. Thus, sucrose transport may be regulated in part by the nature of interactions between the transporter and this interacting partner.This research will attempt to elucidate the functional significance of this protein-protein interaction in sucrose transport. The three primary objectives are as follows.Objective #1. Identify proteins that interact with the ankyrin-like protein. To understand the nature of this interaction, it is important to determine if other proteins might also be brought into contact with the plant sucrose transporter. For instance, if sucrose metabolizing enzymes are part of a complex with the sucrose transporter, then sucrose import may be directly linked to its subsequent metabolism. These experiments will address this possibility.Objective #2. Assess the role of protein-protein interaction on sucrose uptake. If the interaction of the sucrose transporter with the ankyrin-like protein has an impact on sucrose uptake in the plant, then disruption of the ankyrin-like protein function should disrupt sucrose transport in distinct ways. The ankyrin-like protein function will be disrupted using gene-silencing technology and sucrose uptake parameters assessed.Objective #3. Examine the subcellular localization of the sucrose transporter and the ankyrin-like protein. Several important questions are addressed by characterizing the nature of the sucrose transporter:ankyrin-like protein (ST:ALP) interaction in the cell. Does the ST:ALP complex change during cotyledon development? When does the ST:ALP complex form relative to synthesis of the sucrose transporter and the ankryin-like protein? Our mRNA analysis suggests that the ankyrin-like protein accumulates earlier in cotyledon development than does the sucrose transporter and this raises the question as to whether sucrose transporter is bound to the ankyrin-like protein as it is translated. Immunocytochemical analysis of the sucrose transporter, the ankyrin-like protein, and other potential interacting proteins confocal and electron microscopy will address these issues.