24 h after transfection, cells expressing ARL6IP1 SNAP or Halo tag were labeled immediately before imaging with JF646-SNAP-tag ligand or JF646-HaloTag ligand, respectively (gift from L. impartial study, a screen for mutations affecting the distribution of the ER network in dendrites of the PVD neurons of led to the isolation of mutants in CIL-1, which encodes the INPP5K worm orthologue. The mutant phenotype was rescued by expression of wild type, but not of catalytically inactive CIL-1. Our results reveal an unexpected role of an ER localized polyphosphoinositide phosphatase in the fine control of ER network business. Introduction Phosphoinositides are a family of signaling bilayer phospholipids resulting from the reversible phosphorylation of phosphatidylinositol at the 3, 4, and 5 position of the inositol ring. Each of the phosphorylated headgroups recognizes with variable affinity and specificity unique set of protein motifs and domains, thus helping to recruit and regulate cytosolic proteins at membrane interfaces. Via these interactions, CD4 as well as via direct actions on membrane proteins, phosphoinositides play major functions in the control of a variety of physiological processes, including transmission transduction, membrane trafficking, cytoskeleton dynamics, and transport of ion and metabolites across bilayers. Key to this function is the heterogeneous distribution of the different phosphoinositides on different membranes, which is usually achieved and managed through the subcellular targeting of lipid kinases, lipid phosphatases, and lipid transport proteins GW438014A (Di Paolo and De Camilli, 2006; Balla, 2013). Mammalian genomes encode 10 inositol 5-phosphatases. One 5-phosphatase, INPP5A, only functions on soluble inositol polyphosphates, while the other nine have phosphoinositide phosphatase activity (i.e., dephosphorylate the 5 position of lipid-bound inositol polyphosphates), although they can also dephosphorylate soluble inositol polyphosphates (Conduit et al., 2012; Hakim et al., 2012; Pirruccello and De Camilli, 2012). All nine proteins are cytosolic enzymes in which the catalytic module is usually flanked by domains that mediate their subcellular targeting to membranes where they express their catalytic action. Typically, these 5-phosphatases are targeted to membranes distal to the ER, which include the plasma membrane and membranes of the secretory and endocytic pathways, where the bulk of their substrates are localized (Conduit et al., 2012; Hakim et al., 2012; Pirruccello and De Camilli, 2012). One exception is usually INPP5K, a 5-phosphatase GW438014A localized at least in part, on the surface of the ER (Wiradjaja et al., 2001; Gurung et al., 2003). Recombinant full-length INPP5K has 5-phosphatase activity toward PI(4,5)P2 and PI(3,4,5)P3, with marked preference for PI(4,5)P2 (Ijuin et al., 2000; Schmid et al., 2004). However, neither PI(4,5)P2 nor PI(3,4,5)P3 is usually GW438014A thought to be concentrated, or even present, in the ER, raising questions about the physiological function of this localization (Di Paolo and De Camilli, 2006; Balla, 2013). INPP5K, also known as skeletal muscle mass and kidney-enriched inositol 5-phosphatase (SKIP), is usually highly expressed in the developing and adult brain, eye, muscle mass, and kidney (Ijuin et al., 2000). The knockout of INPP5K in mouse results in embryonic lethality (Ijuin et al., 2008). Human biallelic point mutations that impair INPP5Ks phosphatase activity give rise to congenital muscular dystrophy with additional clinical manifestations, including cataracts, intellectual impairments, and short stature (Osborn et al., 2017; Wiessner et al., 2017). Mechanisms of disease, however, remain unclear. Specifically, it is unknown whether the GW438014A ER localization of INPP5K contributes to the disease, as pools of INPP5K not associated with the ER are present. For example, it was shown that upon growth factor activation, a pool of INPP5K can be recruited to the plasma membrane to down-regulate PI(3,4,5)P3 signaling (Gurung et al., 2003). INPP5K has a simple two-domain structure with an N-terminal 5-phosphatase domain name followed by a C-terminal GW438014A SKICH domain name, with no transmembrane regions reported. The closest homologue of INPP5K in yeast, the protein INP54, also localizes at the ER surface, suggesting a highly conserved ER-related function of this enzyme. However, INP54, which lacks the SKICH domain name, is anchored to the ER via a hydrophobic 13-aa C-terminal sequence that is missing in INPP5K (Fig. 1 A; Wiradjaja et al., 2001). How INPP5K is usually targeted to the ER remains unknown. Open in a separate window Physique 1. ER localization of INPP5K.