The mechanisms by which cytosolic proteins reversibly bind the membrane and induce the curvature for membrane trafficking and remodeling remain elusive. The epsin N-terminal homology (ENTH) domain has potent vesicle tubulation activity despite a lack of intrinsic molecular curvature. EPR revealed that the N-terminal ␣-helix penetrates the phosphatidylinositol 4,5-bisphosphate-containing membrane at a unique oblique angle and concomitantly interacts closely with helices from neighboring molecules in an antiparallel orientation. The quantitative fluorescence microscopy showed that the formation of highly ordered ENTH domain complexes beyond a critical size is essential for its vesicle tubulation activity. The mutations that interfere with the formation of large ENTH domain complexes abrogated the vesicle tubulation activity. Furthermore, the same mutations in the intact epsin 1 abolished its endocytic activity in mammalian cells. Collectively, these results show that the ENTH domain facilitates the cellular membrane budding and fission by a novel mechanism that is distinct from that proposed for BAR domains.Cell membranes undergo dynamic structural changes and remodeling during movement, division, and vesicle trafficking (1, 2). In particular, vesicle budding and fusion constantly take place in various cell membranes to maintain communication and transport between membrane-bound compartments (3). Dynamic membrane remodeling involves changes in local membrane curvature (or deformation) that are orchestrated by membrane lipids, integral membrane proteins, and cytoskeletal proteins (4, 5). Recently, several groups of cytosolic proteins that reversibly bind membranes and induce and/or detect different types of membrane curvatures during membrane remodeling have been identified. In particular, cytosolic proteins that are involved in different stages of clathrin-mediated endocytosis have received the most attention (6, 7), and many of them contain either an Ap180 N-terminal homology (ANTH) 2 /epsin N-terminal homology (ENTH) (8, 9) or a Bin-amphiphysin-Rvs (BAR) domain (9 -12). Although ENTH (13) and BAR domains (14 -17) have been reported to have in vitro vesicle-tubulating activities, the exact mechanisms by which these domains induce membrane deformation and larger scale membrane remodeling, especially under physiological conditions, are yet to be elucidated. For BAR domains, their unique intrinsic molecular curvatures have been postulated to be important for membrane deformation through a scaffolding mechanism (4). Also, recent studies have shown that F-BAR domains from FBP17 and CIP4 form highly ordered self-assembly in two-dimensional (18) and three-dimensional (19) crystals and that disruption of intermolecular interactions abrogates their membrane deformation activities. Despite remarkable success in structural characterization of various BAR and ENTH domains, questions still remain as to whether individual domains function by a universal mechanism or by different mechanisms, whether the intact proteins harborin...

Phosphatidylinositol‐4,5‐bisphosphate (PtdIns(4,5)P2), which is a minor component of the plasma membrane, plays important roles in many cellular processes, including signal transduction, exocytosis, endocytosis, and ion channel activation. A method for quantitatively monitoring the PtdIns(4,5)P2 concentration and dynamics in living cells is lacking at present. We have developed a new method of quantifying the PtdIns(4,5)P2 concentration in living cells using a specific PtdIns(4,5)P2 sensor made of the engineered ENTH domain of epsin1 labeled with an environment‐sensitive fluorophore. The PtdIns(4,5)P2 sensor allows robust quantitative determination of spatiotemporal fluctuation of PtdIns(4,5)P2 in giant unilamellar vesicles and mammalian cells by ratiometric analysis. The results demonstrate the spatially heterogeneous distribution of PtdIns(4,5)P2 in the plasma membrane and provide new insight into the fluctuation and dynamics of cellular PtdIns(4,5)P2 in response to various stimuli. Furthermore, this strategy is generally applicable to in situ quantification of other membrane lipids.Supported by NIH GM 68849 and GM76581

Diacylglycerol (DAG) is a signaling lipid that interacts with the C1 domain in various target proteins, including protein kinases C, and Ras guanyl nucleotide‐releasing proteins, thereby recruiting them to the plasma membrane. Although these proteins can all bind DAG, some are known to target other intracellular membranes. To understand the basis of this differential membrane targeting, we explored the possibility that a difference in the acyl chain composition of DAG in the plasma membrane and other membranes may affect the C1‐DAG interaction. To test this notion, we measured the binding of the various C1 domains to DAGs with different sn‐2 acyl groups by fluorescence correlation spectroscopy (FCS). The C1 domains were expressed as an enhanced green fluorescence protein‐fusion protein to enhance protein stability and allow FCS measurements. FCS measurements as well as the molecular dynamics simulation show that the sn‐2 acyl group of DAG has significant and differential effects on the membrane binding properties of C1 domains, which is consistent with the differential subcellular localization of the C1 domains.Supported by NIH GM76581