Vacuolar Targeting Machinery in Plants

MCB-9507030 Raikhel 本项目的长期目标是研究植物液泡蛋白分选的分子机制。 植物液泡分选信号的三种类型已被确定，基于它们在蛋白质中的位置。 在C-末端（CTPP）和N-末端（NTPP）的前肽区域内的信号以及成熟蛋白体内的信号已经被表征。 在不同类型的信号中，似乎没有一个共同的序列基序负责液泡分选，和一个不同的受体是牵连在每个信号的识别。 我们已经表征了植物分泌蛋白中的CTPP信号，并鉴定了引起液泡蛋白分泌的特定突变。 在不同的CTPP之间没有同源性，并且CTPP中的各种突变和缺失被分选机制所容忍。 虽然液泡分选信号和识别这些信号的最可能的受体是独特的，但参与囊泡运输的组分在真核细胞中是常见的。 为此，我们已经从拟南芥中分离出了酵母Pepl 2 p的功能同源物，该同源物是一种被认为参与特定识别泡囊的整合蛋白。我们计划对A. Thaliana Pepl 2 p，并将其用作分离机器其他组件的工具，包括识别液泡分选信号的受体。 为了配合我们的分子和生物化学方法，我们已经分离出几个假定的A。拟南芥突变体与改变的极性分选（AVS）。 1）对aPEP 12 cDNA克隆进行全序列测定，制备抗aPEP 12 p的抗体，并利用细胞器分级分离和电镜技术确定aPEP 12的亚细胞定位。 2)通过将aPEP 12反义转化到A.拟南芥植物表达独立大麦凝集素，孢子素，或植物血凝素，并分析改变的aPepl 2 p的水平上的一个或所有三个不同的可溶性液泡蛋白的分选的影响，通过脉冲追踪标记，然后免疫沉淀和细胞器分级。 3)通过梯度离心鉴定与aPepl 2 p相互作用的组分，并使用aPepl 2 p抗体通过共免疫沉淀纯化它们。 另外的方法将包括亲和层析或化学交联。 相应的基因最终将被克隆。 4)分离拟南芥的avs突变体。 我们将继续筛选分泌含CTPP蛋白的其它突变体，并建立类似的筛选以分离分泌含NTPP蛋白的突变体。 将优先考虑分离交易型突变。 5)表征拟南芥的avs突变体。 将通过脉冲追踪分析和细胞器纯化测试分离的分泌含CTPP蛋白的突变体的分泌或含NTPP蛋白的液泡分选（反之亦然）。 对CTPP或NTPP途径特异的突变体将特别感兴趣，因为这些突变体具有最高的潜力来鉴定植物液泡分选所特有的组分。 然而，影响这两种途径的突变体也可以帮助我们识别重要的共同成分。 这项研究应该提供重要的见解的基本过程中的液泡分类机械，可能是独特的植物或具有广泛的意义。 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选到细胞所需部分的成功率，这可能导致作物改良。 液泡是植物细胞内蛋白质和糖的储存场所。 它也是许多储存蛋白质和糖被分解的位点。 关于后者的功能，空泡有点类似于动物细胞的溶酶体。 在过去的五年里，在确定哺乳动物细胞和酵母中物质被运输到溶酶体的一些细胞机制方面取得了迅速进展。 在这项研究中，这种机制在植物中描述。 植物机械探索开始与植物蛋白质参与液泡交通，也可以工作，让交通到酵母液泡。 描述了这种蛋白质以及与其在植物细胞中相互作用的蛋白质和膜。 通过产生在正确靶向可溶性液泡蛋白中有缺陷的突变体，发现了在正确靶向蛋白质至植物液泡中重要的其他蛋白质和基因。 这增强了我们对植物液泡交通是如何调节的理解。 这项工作有许多最终的应用，包括改变靶向机制的组成部分，以便更有效地将新蛋白质储存和运输到植物液泡中，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. ***