Jcb_201409064 1..11

Phosphoinositides (PIs), the seven metabolites resulting from the phosphorylation of phosphatidylinositol at the 3, 4, and 5 position of the inositol ring, are key regulatory phospholipids localized on the cytosolic leaflets of cellular membranes. Via interactions with proteins mediated by their differentially phos­ phorylated headgroups, they control various aspects of cell phys­ iology including, but not limited to, signaling, membrane traffic and interactions, cytoskeleton dynamics, and lipid homeostasis (De Matteis and Godi, 2004; Di Paolo and De Camilli, 2006; Balla, 2013). Not surprisingly, in view of these pleiotropic ef­ fects, connections between malfunctions of PI metabolism and human disease are becoming progressively evident (McCrea et al., 2008; Ooms et al., 2009; Staiano et al., 2014). The seven PIs are differentially localized on cellular mem­ branes and help define their distinct properties (De Matteis and Godi 2004; Idevall­Hagren and De Camilli, 2014). This implies the occurrence of mechanisms that coordinate membrane trans­ port from one compartment to another with a change in the PI signature. One of the best characterized examples of this conversion is the metabolism of PIs that occurs in the endocytic pathway. Although PI(4,5)P2 is primarily concentrated in the plasma membrane, PI3P is the predominant PI in early endo­ somes (Simonsen et al., 2001; Di Paolo and De Camilli, 2006). Thus, endocytosis, which starts with the PI(4,5)P2­dependent recruitment of endocytic factors to the plasma membrane, is closely coupled to PI(4,5)P2 dephosphorylation (Cremona et al., 1999; Stefan et al., 2002; Milosevic et al., 2011; Nández et al., 2014; Posor et al., 2014), whereas arrival of the endocytic mem­ brane is signaled by the acquisition of PI3P (Patki et al., 1997; Simonsen et al., 2001; Zoncu et al., 2009). The first enzyme to be implicated in the coupling between endocytosis and PI(4,5)P2 dephosphorylation was synapto­ janin 1, a PI phosphatase that is expressed at very high levels in neurons, where it participates in the endocytosis of synaptic vesicles (McPherson et al., 1996; Cremona et al., 1999). Synap­ tojanin 1 and its close homologue synaptojanin 2 (Nemoto et al., 1997) dephosphorylate PI(4,5)P2 via two phosphatase modules The recruitment of inositol phosphatases to endocytic membranes mediates dephosphorylation of PI(4,5)P2, a phosphoinositide concentrated in the plasma membrane, and prevents its accumulation on endosomes. The importance of the conversion of PI(4,5)P2 to PtdIns during endocytosis is demonstrated by the presence of both a 5-phosphatase and a 4-phosphatase (Sac domain) module in the synaptojanins, endocytic PI(4,5)P2 phosphatases conserved from yeast to humans and the only PI(4,5)P2 phosphatases in yeast. OCRL, another 5-phosphatase that couples endocytosis to PI(4,5)P2 dephosphorylation, lacks a Sac domain. Here we show that Sac2/INPP5F is a PI4P phosphatase that colocalizes with OCRL on endocytic membranes, including vesicles formed by clathrin-mediated endocytosis, macropinosomes, and Rab5 endosomes. An OCRL–Sac2/INPP5F interaction could be demonstrated by coimmunoprecipitation and was potentiated by Rab5, whose activity is required to recruit Sac2/INPP5F to endosomes. Sac2/INPP5F and OCRL may cooperate in the sequential dephosphorylation of PI(4,5)P2 at the 5 and 4 position of inositol in a partnership that mimics that of the two phosphatase modules of synaptojanin. Sac2/INPP5F is an inositol 4-phosphatase that functions in the endocytic pathway