Plant Vacuole Biogenesis and Function

哺乳动物细胞、酵母细胞和植物细胞分别含有溶酶体或空泡，它们起溶解或消化区室的作用。然而，植物细胞独特地在液泡中储存许多复杂的代谢产物和蛋白质。植物贮藏液泡与溶胞液泡在膜蛋白质组成和内腔内容物方面存在差异。此外，植物细胞特有的囊泡途径从高尔基体运输到蛋白质储存泡，而网格蛋白包被的囊泡，如在酵母和哺乳动物细胞中，从高尔基体运输到裂解泡。 与酵母液泡相反，植物液泡膜含有非常大量（高达50%的膜蛋白）的液泡膜内蛋白（TIP）。TIPs具有水通道蛋白活性，但大量存在似乎远远超过水运输所需的量。以前已经表明，特定的TIP同种型与特定的液泡功能有关：储存种子型储存蛋白的蛋白质储存液泡由α-加δ-TIP标记;储存不同类型蛋白质、营养储存蛋白的液泡和储存色素的液泡由单独的δ-TIP或δ-加γ-TIP标记;通常存在并含有酸性pH和活性蛋白酶的溶解性空泡仅由γ-TIP标记;当植物细胞饥饿时诱导的自噬空泡似乎仅由α-TIP形式标记。据推测，TIP亚型以某种方式决定了液泡的内部环境，从而决定了液泡的功能。此外，植物细胞必须维持功能上不同的液泡以及各自独立的泡状通路这一事实表明，裂解液泡和储存液泡是不同的细胞器，而不仅仅是一个细胞器的两极。这种区别是非常重要的，因为分离的细胞器假说要求植物细胞保持膜蛋白流动的独立途径，以产生和维持不同的细胞器。此外，这意味着植物细胞具有独特的机制，用于内膜系统的额外细胞器成员的生物合成。由于三种TIP异构体中的每一种都可以在一个细胞内的液泡中单独发现，因此可能存在多达三种不同的植物液泡细胞器。我们的目标是测试这些假设的各个部分。TIP同种型在决定液泡功能中的作用将通过敲除转基因矮牵牛和番茄植物中仅δ-TIP的表达来测试。如果δ-TIP是液泡储存功能所必需的，那么这种敲除植物应该缺乏花瓣色素，并且番茄植物应该不能在叶子和花瓣中积累和储存某些蛋白酶抑制剂，即作为防御分子的营养储存蛋白。此外，将鉴定和克隆与自噬泡相关的α-TIP形式，以确定其与蛋白质储存泡中的α-TIP的差异（如果有的话）。这也可能成为基因敲除实验的目标。第二系列的实验涉及从三个功能不同的液泡纯化的液泡膜的生化特性：蛋白质储存液泡，液泡存储营养储存蛋白，和裂解液泡。从酵母和哺乳动物的研究中，已经出现了这样的概念，即分泌途径内的细胞器的身份是通过每个细胞器膜上存在独特的突触融合蛋白来生化定义的。将对存在于每种液泡类型上的突触融合蛋白进行纯化、克隆和表征，作为功能上不同的液泡作为独特的细胞器假设的测试。 这些研究将为植物细胞建立和维持功能不同的液泡的过程提供新的信息。它们还可能为细胞器生物发生机制提供新的见解。

Mammalian, or yeast and plant cells contain lysosomes or vacuoles, respectively, that function as lytic or digestive compartments. Plant cells uniquely, however, also store many complex metabolic products and proteins in vacuoles. Plant storage vacuoles differ from lytic vacuoles with respect to the protein composition of their membranes as well as their lumenal contents. Additionally, a vesicular pathway unique to plant cells traffics from Golgi to protein storage vacuoles, while clathrin coated vesicles, as in yeast and mammalian cells, traffic from Golgi to lytic vacuoles. In contrast to yeast vacuoles, plant vacuole membranes contain very large amounts (up to 50% of the membrane protein) of Tonoplast Intrisic Proteins (TIPs). TIPs have aquaporin activity, but the large amounts present appear to be in great excess over what would be needed for water transport. It has been shown previously that specific TIP isoforms are associated with specific vacuole functions: Protein storage vacuoles that store seed-type storage proteins are marked by alpha- plus delta-TIP; vacuoles that store different types of proteins, vegetative storage proteins, and vacuoles that store pigments are marked by delta-TIP alone or by delta- plus gamma-TIP; lytic vacuoles that are normally present and contain acidic pH and active proteases are marked by gamma-TIP alone; autophagic vacuoles that are induced when plant cells are starved appear to be marked with a form of alpha-TIP alone. It has been hypothesized that TIP isoforms in some way determine the interior environment of vacuoles and thereby determine vacuole functions. Additionally, the fact that plant cells must maintain functionally distinct vacuoles as well as separate vesicular pathways to each indicate that lytic and storage vacuoles are distinct organelles, not simply two extreme poles of one organelle. This distinction is of substantial importance, because the separate organelle hypothesis requires that plant cells maintain separate pathways for flow of membrane proteins to generate and maintain the different organelles. Additionally, it means that plant cells have unique mechanisms for biogenesis of additional organelle members of the endomembrane system. As each of the three TIP isoforms can be found separately on vacuoles within one cell, it is possible that as many as three distinct plant vacuole organelles may exist. The goal is to test various parts of these hypotheses. The role of TIP isoforms in determining vacuole function will be tested by knocking out expression of only delta-TIP in transgenic petunia and tomato plants. If delta-TIP is required for vacuole storage functions, such knockout plants should lack flower petal pigments, and tomato plants should not be able to accumulate and store certain protease inhibitors, vegetative storage proteins that function as defense molecules, in leaves and flower petals. Additionally, the form of alpha-TIP that is associated with autophagic vacuoles will be identified and cloned to determine its difference, if any, from alpha-TIP in protein storage vacuoles. This also will potentially be a target for knockout experiments. The second series of experiments deals with biochemical characterization of tonoplast membranes purified from three functionally distinct vacuoles: protein storage vacuoles, vacuoles storing vegetative storage proteins, and lytic vacuoles. From yeast and mammalian studies, the concept has emerged that identity of organelles within the secretory pathway is defined biochemically by the presence of a unique syntaxin protein on each organelle membrane. The syntaxins present on each vacuole type will be purified, cloned and characterized as a test of the functionally distinct vacuole as a unique organelle hypothesis. These studies will provide new information about processes by which plant cells establish and maintain functionally distinct vacuoles. They may also provide new insights into mechanisms for organelle biogenesis.