Molecular and Functional Characterization of the Arabidopsis ESCRT-I complex

Chikkaputtaiah, Channakeshavaiah Kolagondanahally (2008). Molecular and Functional Characterization of the Arabidopsis ESCRT-I complex. PhD thesis, Universität zu Köln. Open Access

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Abstract

Recently the Arabidopsis ELCH gene, a homolog of Vps23/ TSG101 and the key component of the plant ESCRT-I complex has been functionally characterized (Spitzer et al, Development, 2006). The elch mutant shows multiple nuclei in various cell types, indicating a role in cytokinesis. VPS28 and VPS37 are other known components of ESCRT-I complex that in combination with elch have synergistic phenotypes in double and triple knock-outs suggesting their involvement in ELCH-dependent regulation of cytokinesis. Cytokinesis regulation is therefore a function of the whole ESCRT system in plants and not a special ESCRT unrelated function of ELCH. The role of ESCRT complexes in yeast cytokinesis is unclear but mutations in one or more ESCRT components of mammals and Arabidopsis induce cytokinesis defects, suggesting that the role of the ESCRT machinery in cytokinesis might be conserved in multicellular organisms. A second Vps23 homolog, VPS23-2 (At5g13860) displays high sequence similarity to ELCH (72%). A dominant-negative VPS23-2 construct phenocopies the elch mutant and expression of VPS23-2 under ELCH promoter can rescue the elch mutant. VPS23-2 is therefore functionally redundant to ELCH. In addition to ELCH and VPS23-2 a third, dicot-specific Vps23 homolog, VPS23-3 (At2g38830) has been identified. It shows a lesser degree of homology (47%) to Arabidopsis ELCH compared to the homology of ELCH to Oryza sativa ELCH (66%) and VPS23-2 (72%). Similar to ELCH, VPS23-3 is ubiquitously expressed, localized on endosomes, binds to ubiquitin with its N-terminal UEV domain and part of a high molecular weight complex in gel filtration/size exclusion chromatography assays suggesting that VPS23-3 is a component of the plant ESCRT system. Surprisingly however, VPS23-3 did not rescue the elch phenotype, when expressed under ELCH promoter control indicating that it has a cellular function different from the other two Vps23 genes. vps23-3 knock-out showed a shift in the molecular weight of the complex on blue-native PAGE and differential interaction pattern in bi-molecular fluorescence complementation and in vitro transcription/ translation assays suggesting that VPS23-3 might serve as an additional, fourth component of the Arabidopsis ESCRT-I complex.

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Thesis (PhD thesis)

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