Vacuolar Targeting Machinery in Plants

本项目的长期目标是研究植物液泡中蛋白质分选的分子机制。基于它们在蛋白质中的位置，已经确定了三种类型的植物液泡分选信号。c端（CTPPs）和n端（NTPPs）的前肽区域内的信号已经被表征，以及成熟蛋白质体内的信号。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类，并且每个信号的识别都涉及一个不同的受体。我们已经鉴定了植物分泌蛋白中的CTPP信号，并确定了引起液泡蛋白分泌的特定突变。不同的CTPP之间没有同源性，分选机制可以容忍CTPP中的各种突变和缺失。虽然液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共同的。为此，我们从拟南芥中分离出酵母Pepl2p的功能同源物，这是一种被认为参与液泡囊泡特异性识别的完整蛋白质。我们计划对拟南芥的Pepl2p进行表征，并将其作为分离该机制其他成分的工具，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，我们分离了几个假定的拟南芥液泡分选（AVS）突变体。在此基础上，提出了以下目标：1)对aPEP12 cDNA克隆进行完整测序，通过细胞器分离和电镜技术，培养抗apep2p抗体，确定其亚细胞位置。2)通过将aPEP12反义定向转化为独立表达大麦凝集素、孢素或植物血凝素的拟南拟南植物，研究appl2p在植物细胞中的功能，并通过脉冲追踪标记、免疫沉淀和细胞器分离分析appl2p水平改变对一种或全部三种可溶性液泡蛋白分选的影响。3)梯度离心鉴定与apep2p相互作用的组分，用aPepl2p抗体共免疫沉淀纯化。其他方法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avs突变体。我们将继续筛选其他分泌含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。将优先考虑事务突变的隔离。5)拟南芥avs突变体的特征。分离的用于分泌含CTPP蛋白的突变体将通过脉冲追踪分析和细胞器纯化测试含ntpp蛋白的分泌或液泡分选（反之亦然）。对于CTPP或NTPP途径的特异性突变体将特别感兴趣，因为它们具有最大的潜力来识别植物液泡分选的独特成分。然而，影响这两种途径的突变也可以帮助我们识别重要的共同成分。该研究将为液泡分选机械的基本过程提供重要的见解，这些过程可能是植物独有的或具有广泛意义的。除了对基础知识的贡献之外，这项工作还可以提高将新基因产物分类到细胞所需部分的成功率，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。关于后一种功能，液泡有点类似于动物细胞溶酶体。在过去的五年中，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快速进展。在本研究中，这种机制在植物中被描述。从参与液泡运输的植物蛋白开始探索植物机制，该植物蛋白也可以允许运输到酵母液泡。描述了这种蛋白质及其在植物细胞中与之相互作用的蛋白质和膜。通过产生可溶性液泡蛋白正确靶向缺陷的突变体，发现了对植物液泡正确靶向蛋白有重要作用的其他蛋白和基因。这增强了我们对植物液泡的交通是如何被调节的理解。这项工作有许多最终的应用，包括改变靶向机制的组成部分，以便更有效地储存和运输新蛋白质到植物液泡中，以生产增强型谷物和种子作物。* * *

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. ***