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

蛋白质通过分泌系统运输的一些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。这种复杂性是某些细胞类型中液泡性质多样的结果。至少观察到了两种不同类型的液泡结构：一种具有裂解功能，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，然后以小运输囊泡的形式通过内膜系统运输。液泡蛋白有信号，可以将它们分类成液泡；如果这种信号被删除，植物细胞就会分泌液泡蛋白。到目前为止，已鉴定的植物液泡分选信号可分为三类：N-末端前肽(NTPP)、C-末端前肽(CTPP)，以及成熟蛋白中特征较少的部分。在豌豆(BP80)和拟南芥(AtELP)中已经发现了一个可能的空泡分选受体，它可能在带有NTPP信号的蛋白质所走的路线上发挥作用。这种可能的受体存在于高尔基体、跨高尔基体网络(TGN)来源的囊泡和泡前室(PVC)。尽管TGN和PVC之间的运输对于将货物蛋白忠实地运送到液泡是至关重要的，但对任何多细胞生物体中的这一过程知之甚少。本项目将重点研究TGN和聚氯乙烯之间的调节运输机制。本项目将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)可能的液泡分选受体的体内功能，(3)PVC，以及(4)将货物运送到该隔室的小泡。虽然拟南芥已经做了很多工作，但到目前为止还没有这种植物的内源液泡蛋白被鉴定出来，也没有可靠的抗体可用来对抗空泡蛋白。Raikhel博士将利用拟南芥EST数据库中的信息，通过从分离的小泡中提纯蛋白质，来制备和表征针对空泡标记的抗体，以分析拟南芥蛋白质到空泡的运输。这些内源标记和她实验室特征良好的异源标记将被用于分析在这项研究过程中创造的野生型和拟南芥突变体的贩运。空泡分选受体AtELP是一个小基因家族的代表。对AtELP的货物特异性的表征仅在体外试验中进行；在体内还没有鉴定出与AtELP结合的蛋白质。一个重要的问题是，AtELP和相关蛋白质是与许多不同的空泡分选信号相互作用，还是只与含有NTPP的货物相互作用。Raikhel博士将尝试使用包括反向遗传学、生物化学和细胞生物学在内的实验策略来回答这个问题。植物中的聚氯乙烯的性质和功能还没有很好的定义。与酵母不同，大多数调节植物高尔基体和PVC之间运输的SNARE蛋白都是由小基因家族代表的。此外，首席调查员发现，在植物中，一些SNARE蛋白的作用与在酵母中不同。实验将解决的问题是，这是植物诱捕的冗余或功能复杂的结果，以及植物PVC的结构和功能多样性的结果。他们还将检查聚氯乙烯的货物和驻留蛋白质，以确定聚氯乙烯中是否只存在携带AtELP的囊泡中的蛋白质，或者聚氯乙烯是否是一个隔间，在那里，采取不同路线的囊泡运送他们的货物。他们将使用免疫沉淀实验，然后用蛋白质组学的方法来获得来自各种囊泡和PVC的货物蛋白质的序列信息。这项研究将为了解多细胞生物体中空泡分选的基本过程提供重要的见解，这可能是植物独有的，具有广泛的意义。除了对基础知识的贡献外，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损害的一个重要方面。

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.