Lipid transfer between cell membrane bilayers at contacts between the endoplasmic reticulum (ER) and other membranes help to maintain membrane lipid homeostasis. We found that two similar ER integral membrane proteins, oxysterol-binding protein (OSBP)–related protein 5 (ORP5) and ORP8, tethered the ER to the plasma membrane (PM) via the interaction of their pleckstrin homology domains with phosphatidylinositol 4-phosphate (PI4P) in this membrane. Their OSBP-related domains (ORDs) harbored either PI4P or phosphatidylserine (PS) and exchanged these lipids between bilayers. Gain- and loss-of-function experiments showed that ORP5 and ORP8 could mediate PI4P/PS counter transport between the ER and the PM, thus delivering PI4P to the ER-localized PI4P phosphatase Sac1 for degradation and PS from the ER to the PM. This exchange helps to control plasma membrane PI4P levels and selectively enrich PS in the PM.

PI4KIIIα is targeted to the plasma membrane via an evolutionarily conserved complex comprised of EFR3 and TTC7 to control PtdIns4P synthesis and the selective enrichment of PtdIns(4,5)P2 in this membrane.

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], a phosphoinositide concentrated predominantly in the plasma membrane, binds endocytic clathrin adaptors, many of their accessory factors, and a variety of actin-regulatory proteins. Here we have used fluorescent fusion proteins and total internal reflection fluorescence microscopy to investigate the effect of acute PI(4,5)P 2 breakdown on the dynamics of endocytic clathrin-coated pit components and of the actin regulatory complex, Arp2/3. PI(4,5)P 2 breakdown was achieved by the inducible recruitment to the plasma membrane of an inositol 5-phosphatase module through the rapamycin/FRB/FKBP system or by treatment with ionomycin. PI(4,5)P 2 depletion resulted in a dramatic loss of clathrin puncta, which correlated with a massive dissociation of endocytic adaptors from the plasma membrane. Remaining clathrin spots at the cell surface had only weak fluorescence and were static over time. Dynamin and the p20 subunit of the Arp2/3 actin regulatory complex, which were concentrated at late-stage clathrincoated pits and in lamellipodia, also dissociated from the plasma membrane, and these changes correlated with an arrest of motility at the cell edge. These findings demonstrate the critical importance of PI(4,5)P 2 in clathrin coat dynamics and Arp2/3-dependent actin regulation.actin ͉ dynamin ͉ epsin ͉ phosphoinositides ͉ rapamycin P hosphorylation-dephosphorylation of the inositol ring of inositol phospholipids plays a major role in cell regulation. Their phosphorylated head groups interact with a variety of cytosolic protein modules and thus mediate the recruitment and regulation of proteins at the membrane-cytosol interface (1-7). A direct interaction with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ], a phosphoinositide concentrated at the plasma membrane, has been demonstrated for all of the endocytic clathrin adaptors and for several of their accessory factors, for the GTPase dynamin, and for many actin regulatory proteins (8-16). The physiological significance of these interactions has been supported by cell biological (9, 15, 17-21) and pharmacological (22) studies in living cells and by forward and reverse genetics. For example, mutations in genes encoding inositol 5-phosphatases, primarily synaptojanin family members, were shown to cause endocytic and actin defects in a variety of organisms (16,(23)(24)(25)(26)(27)(28). However, genetic manipulations that result in long-lasting changes in cellular levels of PI(4,5)P 2 may act indirectly or trigger compensatory mechanisms that can complicate the interpretation of results.Conclusive evidence for the importance of PI(4,5)P 2 in events that occur at the plasma membrane will come from experiments involving the acute and specific manipulation of its levels. Recently, a technique for the rapid, conditional recruitment of proteins to membranes has been used to deliver 5-phosphatase modules to the cell surface (20,(29)(30)(31)(32). In this method, the genes encoding two proteins that can be cross-linked by the add...

The apical and basolateral plasma membrane domains of polarized epithelial cells contain distinct sets of integral membrane proteins. Biosynthetic targeting of proteins to the basolateral plasma membrane is mediated by cytosolic tail determinants, many of which resemble signals involved in the rapid endocytosis or lysosomal targeting. Since these signals are recognized by adaptor proteins, we hypothesized that there could be epithelial-specific adaptors involved in polarized sorting. Here, we report the identification of a novel member of the adaptor medium chain family, named W W1B, which is closely related to the previously described W W1A (79% amino acid sequence identity). Northern blotting and in situ hybridization analyses reveal the specific expression of W W1B mRNA in a subset of polarized epithelial and exocrine cells. Yeast two-hybrid analyses show that W W1B is capable of interacting with generic tyrosine-based sorting signals. These observations suggest that W W1B may be involved in protein sorting events specific to polarized cells.