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.
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