Abstract:
The cytokinesis-specific syntaxin KNOLLE is an essential protein involved in the cell division of somatic Arabidopsis cells. KNOLLE is a key player of the membrane vesicle fusion in the division plane. Detailed information concerning the function of syntaxins is available from animals or yeast which, however, lack KNOLLE orthologous proteins. Thus KNOLLE is a unique syntaxin with its only function during plant cytokinesis. The aim of this thesis was to test the in-vivo significance of in-vitro interactions between Syb4, SNP33, KEULE and KNOLLE. The putative synaptobrevin Syb4 was shown to reside in the ER and its localization was not affected by the vesicle-trafficking inhibitor BFA. In contrast to KNOLLE, Syb4 is constitutively expressed, and the two proteins did not co-localize. These results rule out a direct interaction of Syb4 and KNOLLE and thus question the specificity of in-vitro interaction data. To study the KNOLLE/KEULE interaction, several transgenic plant lines were established. The expression of epitope-tagged KEULE will allow for subcellular protein localization while the expression of a mutated KNOLLE version provides a tool for analysing a postulated interaction site in KNOLLE. To identify new KNOLLE interactors, KNOLLE-containing protein complexes were enriched and analysed by non-denaturing micro-gel electrophoresis. KNOLLE is part of a complex of about 180 kDa. Myc-SNP33 copurified with KNOLLE, which provides direct evidence for interaction of SNP33 and KNOLLE in-vivo.