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

许多转运蛋白的分选和靶向在植物细胞中仍然是难以捉摸的。甚至ER和液泡之间的运输途径在植物中也比例如在酵母中更复杂和更不了解。植物细胞进一步将几种主要活性转运蛋白的不同同种型分选到不同的亚细胞区室中。这种分类是核心的兴趣，因为亚型然后在细胞中承担不同的责任。因此，该项目涉及主要活性转运蛋白-液泡质子转运ATP酶（V-ATP酶）的ER输出。这种酶提供了至少三种同工酶的优势，这些同工酶在亚细胞定位上不同，因此ER驻留同工酶可以与内体同工酶和液泡同工酶进行比较。此外，V-ATP酶的单个亚基似乎能够从内质网绕过高尔基体和内体隔室进入液泡。在酵母中，ER-输出涉及ER出口位点（ERES）、膜整合蛋白的货物受体Erv 14 p和完成分选和囊泡形成的COPII-外壳。这些成分在植物细胞中是可用的，但它们与ER输出的相关性仍不清楚。在本项目中，将在植物细胞中分析V-ATPase的ER输出的ERES依赖性与单个同工酶和运输途径的关系。接下来要解决的关键问题是COP II-外壳在分选V-ATP酶中的功能以及Erv 14 p直系同源物在介导拟南芥外壳和货物蛋白之间的作用。本项目旨在阐明A. thaliana.

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.