Role of the BEACH domain protein SPIRRIG in the context of the ESCRT-System in Arabidopsis thaliana

拟南芥中的SPIRRIG（SPI）基因编码BEACH结构域蛋白，因此被认为在膜转运过程中起作用。四条证据表明SPI参与ESCRT依赖性内体分选过程的调节：1）SPI蛋白与LIP 5和VPS 60相互作用，LIP 5和VPS 60均与SKD 1相互作用; LIP 5调节SKD 1的活性，从而调节ESCRT依赖性蛋白分选的最后一步。2)发现SPI与LIP 5和VPS 60之间的相互作用存在于内体。3)spi、lip 5、vps 60突变体和显性阴性SKD 1系显示液泡的碎裂。4)SDK 1在spi突变体的内体中积累。为了分析SPI在ESCRT依赖的细胞内过程中的作用，我们提出了三条实验路线：1）内膜区室之间的细胞内运输过程的细胞生物学分析，以了解片段化液泡表型的基础。2)蛋白质复合物组成的生化分析和SPI对AtSKD 1的激活。3)通过对来自网骨藻的SPI和LvsA的比较功能分析，对BEACH结构域蛋白和ESCRT功能之间的联系进行进化分析。

The SPIRRIG (SPI) gene in Arabidopsis thaliana encodes a BEACH-domain protein and is therefore thought to play a role in membrane transport processes. Four lines of evidence suggest that SPI is involved in the regulation of ESCRT dependent endosomal sorting processes: 1) SPI protein interacts with LIP5 and VPS60 both of which interact with SKD1; LIP5 regulates the activity of SKD1 and thereby the last step in the ESCRT-dependent protein sorting. 2) The interaction between SPI and LIP5 and VPS60 is found at endosomes. 3) The spi, lip5, vps60 mutants and dominant negative SKD1 lines show fragmentation of the vacuoles. 4) SDK1 accumulates at endosomes in spi mutants. In order to analyze the role of SPI in the context of ESCRT-dependent intracellular processes we propose three lines of experiments: 1) A cell biological analysis of intracellular transport processes between endomembrane compartments in order to understand the basis for the fragmented vacuole phenotype. 2) A biochemical analysis of the composition of the protein complexes and the activation of AtSKD1 by SPI. 3) An evolutionary analysis of the link between BEACH-domain proteins and ESCRT-functions by a comparative functional analysis of SPI and LvsA from Dictyostelium.