Selektiver Transport zur lytischen Vakuole und Degradation von Membranproteinen

植物内体和泡前隔室(EPVCs)被认为是通向溶解液泡的中间隔室。与动物细胞中已知的类似，EPVCs被认为以受体介导的运输方式从高尔基体接收蛋白质，然后将它们输送到液泡。当VSR被送回高尔基体时，预定降解的膜蛋白被认为被分离到这些多囊结构的内部小泡中，然后这些小泡被释放到液泡的腔中(MVB途径)。与动物不同，植物细胞具有不同类型的液泡室，这表明植物EPVCs的分选机制复杂。我们提出了一种结合生化、遗传学和超微结构的方法来：1)分析逆转录体对可溶性液泡蛋白运输以及VSR稳态分布的作用，使用定量蛋白质运输分析结合RNA干扰(RNAi)诱导体内逆转录体亚单位VPS35、Vps29和Vps26编码基因的转录后沉默。2)鉴定植物ESCRT同源物有助于理解负责将膜蛋白分类到MVB管腔小泡中的分子机制。在这里，我们将克隆拟南芥ESCRT基因，以制备植物特异性抗体，作为进行超微结构分析和免疫定位研究的先决条件。为了确定植物ESCRT基因的功能，我们将使用定量蛋白质运输分析结合体内功能互补实验来鉴定可用的拟南芥T-DNA插入突变体。3)确定VSR是否通过MVB途径被翻转。因此，我们将利用脉冲追逐实验结合ESCRT机制瞬时表达的显性负性突变体来分析VSR的半衰期。

Plant endosomal and prevacuolar compartments (EPVCs) are considered to be an intermediate compartment en route to the lytic vacuole. In analogy to what is known from animal cells, it is considered that EPVCs receive proteins from the Golgi- Apparatus in a receptor-mediated transport mode and subsequently deliver them to the vacuole. Whilst VSRs are shuttled back to the Golgi-Apparatus, membrane proteins destined for degradation are thought to be separated into the internal vesicles of these multivesicular structures, which are then discharged into the lumen of the vacuole (MVB pathway). In contrast to animals, plant cells possess different types of vacuolar compartments, suggesting complex sorting mechanisms for plant EPVCs. We propose a combined biochemical, genetical and ultrastructural approach to:1) analyse the role of retromer for transport of soluble vacuolar proteins as well as for the steady-state distribution of VSRs, using quantitative protein transport assays in combination with RNA interference (RNAi) to induce post-transcriptional silencing of genes encoding for retromer subunits Vps35, Vps29 and Vps26 in vivo.2) characterise plant ESCRT homologues as a contribution towards understanding the molecular machinery responsible for sorting of membrane proteins into luminal vesicles of the MVBs. Here, we will clone Arabidopsis ESCRT genes to prepare plant specific antibodies as a prerequisite to conduct ultrastructural analysis and immunolocalization studies. To establish the function of plant ESCRT genes, we will characterize available Arabidopsis T-DNA insertion mutants using quantitative protein transport assays in combination with functional in vivo complementation experiments.3) determine, whether VSRs are turned-over via the MVB pathway. Therefore, we will analyse the half-live time of VSRs using pulse-chase experiments in combination with transiently expressed dominant negative mutants of the ESCRT machinery.