Vacuolar Targeting Machinery in Plants

该项目的长期目标是研究蛋白质对植物液泡进行分选的分子机制。基于它们在蛋白质中的位置，已经识别了三种类型的植物液泡分选信号。C末端(CTPP)和N末端(NTPP)前肽区域内的信号已被鉴定，成熟蛋白质体内的信号也已被鉴定。在不同类型的信号中，似乎没有一个共同的序列基序负责空泡分类，每个信号的识别都涉及一个不同的受体。我们已经确定了植物分泌蛋白中的CTPP信号，并确定了导致液泡蛋白分泌的特定突变。不同的CTPP之间没有同源性，CTPP中的各种突变和缺失是分选机制所能容忍的。虽然空泡分选信号和最有可能识别这些信号的受体是独特的，但参与囊泡运输的成分在真核细胞中是常见的。为此，我们从拟南芥中分离出了酵母Pepl2p的功能同源物，这是一种被认为参与特定识别到达液泡的小泡的完整蛋白。我们计划对A.thaliana Pepl2p进行表征，并将其用作分离该机器的其他组件的工具，包括识别液泡分选信号的受体。为了补充我们的分子和生化方法，我们分离了几个具有改变空泡分选(AVS)的假想突变体。在此基础上，提出了以下目标：1)对aPEP12克隆进行全序列测定，制备抗aPepl2p的抗体，并用它们通过细胞器分级和电子显微镜来确定其在细胞内的位置。2)通过将aPEP12反义导入独立表达大麦L凝集素、孢子胺或植物血凝素的刺参植株，研究aPepl2p在植物细胞中的作用，并通过脉冲追逐标记、免疫沉淀和细胞器分级，分析aPepl2p水平的变化对三种不同可溶性液泡蛋白中的一种或全部分选的影响。3)通过梯度离心法鉴定与aPepl2p相互作用的成分，并使用aPepl 2p抗体进行免疫共沉淀法纯化。其他方法将包括亲和层析或化学交联法。相应的基因最终将被克隆。4)分离拟南芥的avs突变体。我们将继续筛选更多分泌含CTPP蛋白的突变体，并建立类似的筛选来分离分泌含NTPP蛋白的突变体。重点将放在分离交易突变上。5)拟南芥avs突变体的特征。分离出的分泌含CTPP蛋白的突变体将通过脉冲追逐分析和细胞器纯化来测试含NTPP蛋白的分泌或空泡分离(反之亦然)。CTPP或NTPP途径的突变体将特别引起人们的兴趣，因为这些突变体具有最高的潜力来识别植物液泡分离所特有的成分。然而，影响这两条途径的突变也可以帮助我们识别重要的共同成分。这项研究应该为液泡分拣机械的基本过程提供重要的见解，这些过程可能是植物所独有的，也可能具有广泛的意义。除了对基础知识的贡献外，这项工作还可以提高将新基因产物分类到细胞所需部位的成功率，这可能会导致作物改良。液泡是植物细胞内储存蛋白质和糖的地方。它也是许多储存蛋白质和糖被分解的地方。就后一种功能而言，液泡与动物细胞溶酶体有些相似。在过去的五年里，在鉴定一些细胞机制方面取得了快速的进展，通过这些机制，材料被运送到哺乳动物细胞和酵母中的线粒体内。在这项研究中，这种机械是在植物中描述的。从一种参与液泡运输的植物蛋白质开始探索植物机制，这种蛋白质也可以允许运输到酵母液泡。描述了这种蛋白质以及它在植物细胞中与之相互作用的蛋白质和膜。通过产生在正确定位可溶性液泡蛋白方面有缺陷的突变体，可以发现其他在将蛋白质正确定位到植物液泡中起重要作用的蛋白质和基因。这增强了我们对进入植物液泡的流量是如何被调节的理解。这项工作有许多最终的应用，包括改变靶向机械的组件，以便更有效地将新蛋白质储存和运输到植物液泡，以生产增强的谷物和种子作物。***

MCB-9507030 Raikhel The long-term goal of this project is to investigate the molecular mechanisms of protein sorting to plant vacuoles. Three types of plant vacuolar sorting signals have been identified, based on their location within proteins. Signals within the propeptide regions at the C-terminus (CTPPs) and N-terminus (NTPPs) have been characterized as well as signals within the body of mature proteins. Among the different types of signals, there does not appear to be a common sequence motif responsible for vacuolar sorting, and a distinct receptor is implicated in the recognition of each signal. We have characterized the CTPP signals in plant secretory proteins and identified specific mutations that cause secretion of vacuolar proteins. There is no homology between different CTPPs, and various mutations and deletions in CTPP are tolerated by the sorting machinery. Although vacuolar sorting signals and most likely receptors that recognize these signals are unique, the components involved in vesicle transport are common among eukaryotic cells. To this end we have isolated a functional homolog of yeast Pepl2p from Arabidopsis thaliana, an integral protein thought to be involved in the specific recognition of vesicles destined to the vacuole. We plan to characterize the A. thaliana Pepl2p and use it as a tool to isolate other components of the machinery, including the receptors that recognize vacuolar sorting signals. To compliment our molecular and biochemical approaches we have isolated several putative A. thaliana mutants with Altered Vacuolar Sorting (AVS). Based on this the following objectives are proposed: 1) Completely sequence the aPEP12 cDNA clone, raise antibodies against aPepl2p, and use them to determine the subcellular location, by organellar fractionation and electron microscopy. 2) Study the function of aPepl2p in plant cells by transforming aPEP12 in the antisense orientation in to A. thaliana plants expressing independently barley l ectin, sporamin, or phytohemagglutinin, and analyze the effect of altered levels of aPepl2p on sorting of either one or all three different soluble vacuolar proteins by pulse-chase labeling followed by immunoprecipitation and organellar fractionation. 3) Identify components that interact with aPepl2p by gradient centrifugation and purify them by co-immunoprecipitation using aPepl 2p antibodies. Additional approaches will include affinity chromatography or chemical cross-linking. Corresponding genes will eventually be cloned. 4) Isolate avs mutants of Arabidopsis. We shall continue to screen for additional mutants that secrete CTPP-containing proteins and set up a similar screen to isolate mutants that secrete NTPP- containing proteins. Priority will be placed on the isolation of transacting mutations. 5) Characterize avs mutants of Arabidopsis. The mutants isolated for secretion of CTPP containing proteins will be tested for secretion or vacuolar sorting of NTPP-containing proteins (and vice versa) by pulse-chase analysis and organellar purification. Mutantsspecific for either the CTPP or the NTPP pathways will be of particular interest because these have the highest potential to identify components unique to plant vacuolar sorting. However, mutants that affect both pathways could also help us to identify important common components. The research should provide important insights into the fundamental processes of the vacuolar sorting machinery that may be unique to plants or of broad significance. Beyond its contribution to basic knowledge, this work could improve the success rate of sorting novel gene products to desired parts of the cell which might lead to crop improvement. %%% The vacuole is the site of storage of protein and sugars within the plant cell. It is also the site at which many of these storage proteins and sugars are broken down. With regard to this latter function, the vacuole is somewhat similar to the animal cell lysosome. Over the past five years, there has been rapid progress in identifying some of the cellular machinery by which material is trafficked to the iysosome in mammalian cells and yeast. In this study, this machinery is described in plants. The plant machinery is explored starting with a plant protein involved in vacuole traffic which can also work to allow traffic to the yeast vacuole. This protein and the protein and membranes with which it interacts in the plant cell are described. Other proteins and genes important in correctly targeting proteins to plant vacuoles are discovered through generating mutants defective in correct targeting of soluble vacuolar proteins. This enhances our understanding of how traffic to the plant vacuole is regulated. There are many eventual applications of this work, including altering the components of the targeting machinery in order to get more efficient storage and transport of novel proteins to the plant vacuole for the production of enhanced grain and seed crops. ***