The Arabidopsis elch mutant reveals functions of an ESCRT component in cytokinesis

The Endosomal Sorting Complex Required for Transport (ESCRT) regulates important functions in the secretory system of yeast and animals. The main responsibilities that have been described so far are sorting of biosynthetic cargo and receptor-downregulation. The scope of my PhD thesis was the analysis of the Arabidopsis ELCH protein that is similar to Vps23p and TSG101. These proteins represent the core components of ESCRT I complex in yeast and animals. I could show that ubiquitin binding and complex formation is conserved between Arabidopsis, yeast and animals, supporting the idea that ESCRT mediated protein sorting is a general strategy in eukaryotic organisms. New ELCH/ESCRT-I interacting proteins were isolated by immunoprecipitation and subsequent mass spectrometry. By this approach a plant specific protein containing a UBiquitin Associated (UBA) domain and several subunits of the vacuolar (H+)-ATPase were identified. The VHA-a3 subunit of the vacuolar (H+)-ATPase was analysed in more detail for ubiquitin modifications because mono-ubiquitination constitutes the sorting signal for the ESCRT pathway. Two colour western analysis showed that VHA-a3 is mono-ubiquitinated indicating that VHA-a3 might be a target of ELCH/ESCRT-I. Similar to the ESCRT pathway the V-ATPase is involved in the sorting of biosynthetic cargo and receptor-downregulation in yeast. No interaction between the ESCRT pathway and the V-ATPase has been reported so far. A T-DNA mutation in the ELCH gene of Arabidopsis results in multiple nuclei in a minority of epidermal cells. As multinucleated cells can be an indication for a cell division defect, trichomes, pavement cells and stomata were examined in respect to incomplete cell walls. Cell wall stubs were observed in pavement cells and stomata but not in trichomes. Similar defects have not been observed in yeast vps23 but the multinucleated phenotype of elch resembles the phenotype observed in TSG101 mutant cell lines. Furthermore cytokinesis defects are reported in Arabidopsis mutants lacking the VHA-E subunit of the V-ATPase. Plants mutant for VHA-E are embryonic lethal and display incomplete cell walls, multiple nuclei and aberrant vacuoles (Strompen et al., 2005). Although mutations in ELCH, TSG101 and VHA-E cause a similar phenotype only vague ideas exist why compromising the ESCRT pathway or the V-ATPase lead to cell division defects. A cue was provided by genetic analysis, which suggests that ELCH influences cell division by regulating microtubules. This is apparent because a double mutant with tubulin-folding cofactor a (tfc-a) shows a strong synergistic phenotype. Cell wall development during plant cell division depends heavily on a plant specific structure, the phragmoplast. The secretory system and microtubules are the main components of the phragmoplast. Therefore it seems reasonable to assume that subtle protein sorting defects, mislocalization of membranes or misregulation of microtubules can lead to the observed cell division defect.