Vesicle Trafficking From the Trans-Golgi Network to Prevacuolar Compartment in Arabidopsis

一些蛋白质通过分泌系统运输的机制在所有真核生物中都是保守的；然而，植物具有高度复杂的液泡靶向机制。这种复杂性是由于某些细胞类型中液泡的多样性造成的。至少有两种不同类型的液泡结构被观察到：一种具有溶解功能，另一种具有储存功能。分泌途径的蛋白质通常首先插入内质网，然后在小运输囊泡中通过膜系统运输。液泡蛋白具有将其分类到液泡的信号；如果这些信号被删除，液泡蛋白就会从植物细胞中分泌出来。目前已知的植物液泡分选信号可分为三类：n端前肽（NTPPs）、c端前肽（CTPPs）和成熟蛋白中特征较少的部分。在豌豆（BP80）和拟南芥（AtELP）中发现了一个假定的液泡分选受体，它可能在具有NTPP信号的蛋白质所采取的途径中起作用。这种假定的受体存在于高尔基体、反式高尔基网络（TGN）衍生的囊泡和泡前室（PVC）中。尽管TGN和PVC之间的运输对于将货物蛋白忠实地递送到液泡至关重要，但在任何多细胞生物中对这一过程知之甚少。该项目将重点研究TGN和PVC之间的转运机制。在这个项目中进行的实验旨在进一步研究(1)植物中NTPP途径的机制和多样性，(2)假定的液泡分选受体的体内功能，(3)PVC，以及(4)向该隔间运送货物的囊泡。虽然对拟南芥进行了大量的研究，但尚未发现该植物的内源性空泡蛋白，也没有针对空泡蛋白的可靠抗体。Raikhel博士将利用拟南芥EST数据库中的信息和从分离的囊泡中纯化蛋白质，制备和表征针对液泡标记的抗体，分析拟南芥蛋白质到液泡的运输。这些内源标记和她的实验室的鉴定良好的异源标记将用于分析野生型和拟南芥突变体在本研究过程中产生的贩运。假设的液泡分选受体，atlp，是一个小基因家族的代表。表征atlp的货物特异性只在体外试验中完成；在体内没有发现与atlp结合的蛋白。一个重要的问题是，atlp和相关蛋白是与许多不同的液泡分选信号相互作用，还是只与含有ntpp的货物相互作用。Raikhel博士将尝试用包括反向遗传学、生物化学和细胞生物学在内的实验策略来回答这个问题。聚氯乙烯在植物中的性质和作用还不明确。与酵母不同的是，植物中大多数介导高尔基体和聚氯乙烯之间运输的SNARE蛋白都是由小基因家族代表的。此外，首席研究员还发现，在植物中，一些SNARE蛋白的作用与酵母不同。实验将解决这是否是植物SNAREs冗余或功能复杂性的结果，以及植物pvc的结构和功能多样性的问题。他们还将检查聚氯乙烯的货物和驻留蛋白质，以确定是否只有在携带atlp的囊泡中发现的蛋白质存在于聚氯乙烯中，或者聚氯乙烯是否是一个囊泡通过不同途径运送货物的隔间。他们将使用免疫沉淀实验，然后使用蛋白质组学方法获得来自各种囊泡和聚氯乙烯的货物蛋白的序列信息。该研究将为多细胞生物液泡分选的基本过程提供重要的见解，这可能是植物所特有的，具有广泛的意义。除了对基础知识的贡献之外，这项工作还将提高将新基因产物分类到细胞所需部分的成功率，这是作物设计和生物伤害的一个重要方面。

Some mechanisms of protein trafficking through the secretory system are conserved among all eukaryotes; nevertheless, plants possess a highly complex vacuolar targeting machinery. The complexity is the result of the diverse nature of vacuoles in some cell types. At least two distinct types of vacuolar structure have been observed: one with a lytic and one with a storage function. Proteins of the secretory pathway are usually first inserted into the endoplasmic reticulum before being transported through the endomembrane system in small transport vesicles. Vacuolar proteins have signals that sort them to vacuoles; if such signals are deleted, vacuolar proteins are instead secreted from the plant cell. The plant vacuolar sorting signals identified to date fall into three classes: N-terminal propeptides (NTPPs), C-terminal propeptides (CTPPs), and much less characterized portions of mature proteins. A putative vacuolar sorting receptor has been identified in pea (BP80) and in Arabidopsis (AtELP), and it likely functions in the route taken by the proteins with NTPP signals. This putative receptor has been found in the Golgi, trans-Golgi network (TGN)-derived vesicles, and in the prevacuolar compartment (PVC). Although transport between the TGN and PVC is critical for faithful delivery of cargo proteins to the vacuole, very little is known about this process in any multicellular organism. This project will focus on mechanisms that mediate transport between the TGN and PVC. Experiments to be carried out in this project aim at investigating further (1) the mechanisms and diversity of NTPP pathway in plants, (2) the in vivo function of the putative vacuolar sorting receptor, (3) the PVC, and (4) vesicles that deliver cargo to that compartment. Although much work has been done with Arabidopsis, no endogenous vacuolar proteins from this plant have as yet been characterized and no reliable antibodies against vacuolar proteins are available. Dr. Raikhel will prepare and characterize antibodies against vacuolar markers to analyze transport of Arabidopsis proteins to the vacuoles using information available in the Arabidopsis EST database and by purifying proteins from isolated vesicles. These endogenous markers and her laboratory's well-characterized heterologous markers will be used to analyze trafficking in both wild-type and Arabidopsis mutants created in the course of this research. The putative vacuolar sorting receptor, AtELP, is a representative of a small gene family. Characterization of the cargo specificity of AtELP has been done only in in vitro assays; no proteins that bind AtELP in vivo have been identified. One important question is whether AtELP and related proteins interact with a number of different vacuolar sorting signals or only with NTPP-containing cargo. Dr. Raikhel will try to answer this question using experimental strategies that include reverse genetics, biochemistry, and cell biology. The nature and function of the PVC in plants is not well defined. Unlike in yeast, most SNARE proteins that mediate trafficking between the Golgi and PVC in plants are represented by small gene families. In addition, the Principal Investigator has found that in plants, some SNARE proteins have different roles than in yeast. Experiments will address the question of whether this is a result of redundancy or functional complexity of plant SNAREs, and structural and functional diversity of the plant PVCs. They will also examine the cargo and resident proteins of the PVC to see whether only proteins found in the AtELP-carrying vesicles are present in the PVC, or whether the PVC is a compartment where vesicles taking different routes deliver their cargo. They will use immunoprecipitation experiments followed by a proteomic approach to obtain sequence information of cargo proteins from various vesicles and the PVC.This research will provide important insights into the fundamental processes of vacuolar sorting in multicellular organisms that may be unique to plants and are of broad significance. Beyond its contribution to basic knowledge, this work will improve the success rate of sorting novel gene products to desired parts of the cell, an important aspect of crop design and biopharming.