Park, M.; Droste-Borel, I.; Macek, B.; Juergens, G.

In plant cytokinesis, the partitioning membrane is made by homotypic fusion of secretory vesicles, progressing in a centre-to-periphery direction. In Arabidopsis, this process is mediated by a cytokinesis-specific fusion machinery involving Qa-SNARE KNOLLE which is made during G2/M phase and degraded at the end of cytokinesis. Here we analyse how the turnover of KNOLLE protein is regulated. KNOLLE is ubiquitinated, which is best detected after combined treatment with inhibitors of endocytosis and de-ubiquitination. Site-directed mutagenesis of three clustered lysine residues prevented ubiquitination and internalisation, resulting in stable accumulation of KNOLLE at the plasma membrane in all cells of the seedling root. This is in stark contrast to the transient accumulation of wild-type KNOLLE in dividing cells only. Partial-substitution mutant lines revealed redundancy of lysine residues in both KNOLLE ubiquitination and turnover. KNOLLE ubiquitination resulted in K63-linked ubiquitin chains known to be involved in endocytosis whereas K48-linked chains were not detected. To explore the spatio-temporal conditions, we analysed KNOLLE ubiquitination in cis-SNARE and trans-SNARE complexes during membrane traffic and cell-plate formation. Our findings suggest that KNOLLE protein turnover is caused by a ubiquitination process that depends on successful membrane fusion generating the cell plate.