Dynamic association of the PI3P-interacting Mon1-Ccz1 GEF with vacuoles is controlled through its phosphorylation by the type 1 casein kinase Yck3

Abstract

Maturation of organelles in the endolysosomal pathway requires exchange of the early endosomal GTPase Rab5/Vps21 for the late endosomal Rab7/Ypt7. The Rab exchange depends on the guanine nucleotide exchange factor activity of the Mon1-Ccz1 heterodimer for Ypt7. Here we investigate vacuole binding and recycling of Mon1-Ccz1. We find that Mon1-Ccz1 is absent on vacuoles lacking the phosphatidic acid phosphatase Pah1, which also lack Ypt7, the phosphatidylinositol 3-kinase Vps34, and the lipid phosphatidylinositol 3-phosphate (PI3P). Interaction of Mon1-Ccz1 with wild-type vacuoles requires PI3P, as shown in competition experiments. We also find that Mon1 is released from vacuoles during the fusion reaction and its release requires its phosphorylation by the type 1 casein kinase Yck3. In contrast, Mon1 is retained on vacuoles lacking Yck3 or when Mon1 phosphorylation sites are mutated. Phosphorylation and release of Mon1 is restored with addition of recombinant Yck3. Together the results show that Mon1 is recruited to endosomes and vacuoles by PI3P and, likely after activating Ypt7, is phosphorylated and released from vacuoles for recycling.