Yck3 casein kinase-mediated phosphorylation determines Ivy1 localization and function at endosomes and vacuole.

The casein kinase Yck3 is a central regulator at the vacuole that phosphorylates several proteins involved in membrane trafficking. Here, we set out to identify novel substrates. We localized endogenously tagged Yck3 not only at the vacuole, but also on endosomes. To disable Yck3 function, we generated a kinase-deficient mutant and thus identified the I-BAR-protein Ivy1 as a novel Yck3 substrate. Ivy1 localizes to both endosomes and vacuoles, and Yck3 controls this localization. A phosphomimetic Ivy1-SD mutant was found primarily on vacuoles, its non-phosphorylatable SA variant strongly localized to endosomes, similar to what is observed upon deletion of Yck3. In vitro analysis revealed that Yck3-mediated phosphorylation strongly promoted Ivy1 recruitment to liposomes carrying Rab7-like Ypt7. Modelling of Ivy1 with Ypt7 identified binding sites for Ypt7 and a positively charged patch, which are both required for Ivy1 localization. Strikingly, Ivy1 mutations in either site resulted in more cells with multilobed vacuoles, suggesting a partial defect in its membrane biogenesis. Our data thus indicate that Yck3-mediated phosphorylation controls both localization and function of Ivy1 in endolysosomal biogenesis.