Growing evidence has indicated that inositol metabolism plays pivotal roles in membrane trafficking processes within cells, in addition to its well established role in intracellular signaling pathways. An emerging concept is that various phosphoinositide stereoisomers, with phosphate groups at different positions of the inositol ring, are recognized by specific proteins that are involved in generation of membrane vesicles and in their docking to acceptor compartments. Thus, changes in phosphoinositide levels in a spatially and temporally regulated manner are means by which membrane trafficking events are controlled. These mechanisms seem to underlie the requirements for several enzymes and proteins responsible for synthesis, intracellular transport and breakdown of distinct phosphoinositides in specific membrane trafficking steps (Roth and Sternweis, 1997; Martin, 1998, and references therein).
We have recently identified a neuronal inositol polyphosphate 5′-phosphatase, synaptojanin 1 (McPherson et al., 1994a, 1996) and a more broadly expressed form, synaptojanin 2 (Nemoto et al., 1997). Synaptojanins dephosphorylate inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate at the D-5 position of the inositol ring in vitro (McPherson et al., 1996; Nemoto et al., 1997; Woscholski et al., 1997) and define a subfamily of inositol 5′-phosphatases with a unique three-domain structure: an N-terminal domain homologous to Saccharomyces cerevisiae Sac1p (Cleves et al., 1989; Novick et al., 1989), a central inositol 5′-phosphatase domain and a C-terminal proline-rich domain. Synaptojanin 1 and dynamin 1 are highly enriched in nerve terminals, where they are localized on coated endocytic intermediates (McPherson et al., 1994b; Haffner et al., 1997). Both proteins undergo dephosphorylation in response to nerve terminal depolarization (Robinson et al., 1993; McPherson et al., 1994b; Bauerfeind et al., 1997), and it has been shown recently that such depolarization-induced dephosphorylation promotes the assembly of several proteins for compensatory endocytosis in nerve terminals (Slepnev et al., 1998). Synaptojanin 1 and dynamin 1 specifically interact with the SH3 domains of Grb2 (Gout et al., 1993; McPherson et al., 1994a), amphiphysin 1 and 2 (David et al., 1996; Micheva et al., 1997; Ramjaun et al., 1997), members of the SH3p4/8/13 protein family (endophilins) (de Heuvel et al., 1997; Ringstad et al., 1997), intersectin (Yamabhai et al., 1998) and syndapin I (Qualmann et al., 1999). These properties suggest that synaptojanin 1, together with dynamin 1, functions in the retrieval of the synaptic vesicle membrane by clathrin-coated vesicles. Inositol phospholipids have been reported to bind to several proteins implicated in this process with high affinity, and in some cases modulate their biochemical properties. Examples include the α-subunit of clathrin assembly protein complex AP2, the neuron-specific clathrin assembly protein AP180, the C2B domain of synaptotagmin and the pleckstrin homology domain of dynamin 1 (reviewed in Cremona and De Camilli, 1997). Furthermore, we and others have recently shown that deletion of yeast synaptojanin-like genes causes a severe defect in receptor-mediated and fluid phase endocytosis (Singer-Krüger et al., 1998; Stolz et al., 1998). These results, together with those of Luo and Chang (1997), provided the first direct evidence for a role for synaptojanins in endocytic membrane trafficking in S.cerevisiae.
Synaptojanin 2 undergoes extensive alternative splicing in the C-terminal proline-rich region to generate multiple isoforms (Khvotchev and Südhof, 1998; Seet et al., 1998). Our initial characterization of synaptojanin 2 (now referred to as synaptojanin 2A) showed that synaptojanin 2A is associated predominantly with the particulate fraction, while synaptojanin 1 is localized mainly in the soluble fraction in transfected Chinese hamster ovary (CHO) cells, and that the different protein–protein interactions in their proline-rich region are likely to direct synaptojanin 1 and 2A to different subcellular compartments (Nemoto et al., 1997). To explore the biochemical basis of the previous findings, in this study we performed a yeast two-hybrid screening for synaptojanin 2A-specific interacting proteins and we have identified a novel mitochondrial outer membrane protein with a single PDZ domain, OMP25. We show that the PDZ domain of OMP25 binds to the unique motif in the C-terminal sequence specific to synaptojanin 2A among several alternatively spliced forms of synaptojanin 2. By this interaction, OMP25 mediates the recruitment of synaptojanin 2A to mitochondria, where it causes an alteration of morphology of the mitochondrial network. OMP25 represents the first identified component in mammalian mitochondria that appears to play a role in the proper distribution of mitochondria. Our observations implicate the specific pools of inositol phospholipids in regulation of the dynamics of intracellular organelles and extend the roles of inositol metabolism in cell physiology.