Dynamic association of the PI 3 P-interacting Mon 1-Ccz 1 GEF with vacuoles is controlled through its phosphorylation by the type 1 casein kinase Yck 3

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. INTRODUCTION Eukaryotic cells are compartmentalized by membrane-bound organelles, leading to the specific localization of proteins and enzymatic reactions. Cargo is packaged into vesicles at donor organelles and transported to acceptor membranes, culminating in a fusion event that is regulated by organelle-specific Rab GTPases, their effectors, and soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) proteins. It was discovered that organelles can mature from early to late stages, characterized in part by sequential recruitment and activation of Rab proteins (Rivera-Molina and Novick, 2009). In a general model it is now believed that Rab GTPase guanine nucleotide exchange factors (GEFs) regulate Rab delivery to specific membranes (Gerondopoulos et al., 2012). In the endocytic pathway, early endosomes recruit and activate Rab5/ Ypt21 through the function of the GEF Vps9/Rabex-5 (Hama et al., 1999; Esters et al., 2001). GTP-bound Rab5 is the active form of the protein and can recruit early endosome–specific effectors, including the tethering factor EEA1 and rabaptin-5 (Stenmark et al., 1995; Christoforidis et al., 1999; McBride et al., 1999). Rabaptin-5 inhibits the GTPase activity of Rab5, prolonging the “on” state of Rab5 (Rybin et al., 1996). In Caenorhabditis elegans, Rab5-positive endosomes recruit SAND-1/Mon1, the GEF for the late endosome and lysosome–specific Rab7. Recruitment of SAND-1 occurs in a phosphatidylinositol 3-phosphate (PI3P)–dependent manner (Poteryaev et al., 2010). SAND-1 physically interacts with the Rab5 GEF Rabex-5, leading to its displacement. Subsequently, Rab5 hydrolyzes GTP to become the inactive GDP-bound form, which is then lost from the endosome. However, the constitutively active Rab5Q79L mutant is resistant to SAND-1–mediated inactivation, indicating that Monitoring Editor Patrick J. Brennwald University of North Carolina Received: Aug 12, 2013 Revised: Mar 5, 2014 Accepted: Mar 7, 2014 This article was published online ahead of print in MBoC in Press (http://www .molbiolcell.org/cgi/doi/10.1091/mbc.E13-08-0460) on March 12, 2014. *These authors contributed equally to this work. Address correspondence to: Rutilio A. Fratti (rfratti@illinois.edu). © 2014 Lawrence, Brown, Flood, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/ licenses/by-nc-sa/3.0). “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society of Cell Biology. Abbreviations used: CORVET, class C core vacuole/endosome tethering; GAP, GTPase-activating protein; GEF, guanine nucleotide exchange factor; HOPS, homotypic fusion and vacuole protein-sorting; PI, phosphatidylinositol; PI3P, phosphatidylinositol 3-phosphate; SNARE, soluble N-ethylmaleimide–sensitive factor attachment protein receptor; YPD, yeast extract/peptone/dextrose.