Analysis of ER-Export of vacuolar transporter using the example of the V-ATPase in Arabidopsis thaliana

在植物细胞中，许多转运体的分类和靶向仍然是难以捉摸的。甚至内质网和液泡之间的运输路线在植物中也比在酵母中更复杂，更不为人所知。植物细胞进一步将几种初级活性转运蛋白的不同同工型分类到不同的亚细胞区室中。这种分选是人们感兴趣的中心问题，因为同种异构体在细胞中承担着不同的职责。因此，该项目涉及主要活性转运体，液泡质子易位atp酶（v - atp酶）的er输出。该酶具有至少三种不同亚细胞定位的同工酶的优势，因此内质网同工酶可以与内体和空泡同工酶进行比较。此外，v - atp酶的单个亚基似乎能够绕过高尔基体和内体室，从内质网进入液泡。在酵母中，内质网输出包括内质网出口位点（ERES）、膜整合蛋白的货物受体Erv14p和完成分选和囊泡形成的copii包被。这些成分在植物细胞中是可用的，但它们与er输出的相关性尚不清楚。在本项目中，将从单个同工酶和运输途径的角度分析植物细胞中v - atp酶er输出的es依赖性。接下来要解决的关键问题是拟南芥中COP ii -外壳在v - atp酶分类中的功能以及Erv14p同源物在外壳和货物蛋白之间的介导作用。本项目旨在阐明拟南芥转运蛋白er输出的机制。

The sorting and targeting of many transporters remains elusive in the plant cell. Even the transport routes between ER and vacuole are more complex and less understood in plants than e.g. in yeast. Plant cells further sort different isoforms of several primary active transporters into different subcellular compartments. This sorting is of central interest since the isoforms then take on different responsibilities in the cell. Therefore, the project deals with the ER-export of a primary active transporter, the vacuolar proton translocating ATPase (V-ATPase). This enzyme offers the advantage of at least three isoenzymes that differ in their subcellular localization, so that an ER-resident isoenzyme can be compared with an endosomal and a vacuolar isoenzyme. Furthermore, individual subunits of the V-ATPase seem to be capable to take an alternative route from the ER to the vacuole bypassing the Golgi and endosomal compartments. In yeast, the ER-export involves ER exit sites (ERES), the cargo receptor Erv14p for membrane integral proteins, and the COPII-coat for completing sorting and vesicle formation. These components are available in the plant cell, but their relevance for ER-export is still unclear. In the present project, the ERES-dependency of V-ATPase’s ER-export will be analyzed in the plant cell with respect to the individual isoenzymes and transport routes. The next key-questions that will be addressed are the function of the COP II-coat in sorting the V-ATPase and the role of an Erv14p ortholog in mediating between coat and cargo proteins in Arabidopsis thaliana. The project aims at elucidating the mechanism of ER-export of transport proteins in A. thaliana.