Cell Vol 111 143 146 October 18 2002 Copyright 2002 by Cell Press Endocytosis Driving Membranes around the Bend James H Hurley1 3 and Beverly Wendland2 3 Laboratory of Molecular Biology National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda Maryland 20892 2 Department of Biology Johns Hopkins University 3400 North Charles Street Baltimore Maryland 21218 1 When a nascent vesicle buds the membrane must curve Several mechanisms have been proposed for curvature creation or stabilization Structural analysis of the ENTH domain of the endocytic protein epsin has suggested a new mechanism in which the ENTH domain pushes its way into membranes thus bending them into shape The plasma membrane of cells presents a barrier to the entry of large water soluble particles and nutrients such as iron folate and LDL cholesterol This barrier is overcome through endocytosis the process by which portions of the plasma membrane and extracellular fluid are taken up into a cell Endocytosis is also critical for many physiological responses for instance downregulation of certain signaling receptors Endocytosis can occur via several distinct pathways The best characterized pathway depends upon the cytosolic protein complex clathrin along with its accessory factors reviewed in Brodsky et al 2001 Kirchhausen 2000 The process begins by gathering together plasma membrane proteins and lipids through interactions with cytosolic adaptors and accessory factors Some of these cytosolic factors bind to clathrin and stimulate its polymerization into a spherical basket of pentagons and hexagons As the endocytic vesicle forms the plasma membrane and associated proteins and adjacent extracellular fluid are pulled inward into the cytosol Upon scission the clathrin coated vesicle is released into the cytoplasm Figure 1 Clathrin accessory factors generally exhibit multiple protein interaction domains that allow them to form a web of contacts Clathrin coated vesicles are not highly enriched for accessory factors thus these factors are not structural but rather perform regulatory or catalytic functions at specific stages in endocytosis One accessory factor epsin was discovered in 1998 by virtue of its binding to another accessory factor eps15 Chen et al 1998 Epsin has become a focus of interest due to its interactions with membranes accessory proteins clathrin ubiquitin and possibly certain cargo proteins Wendland 2002 Membranes Get Bent out of Shape How do changes in curvature of the membrane bilayer occur during bud formation and resolution of the coated 3 Correspondence james hurley nih gov J H H bwendland jhu edu B W Minireview pit into a vesicle The process of clathrin polymerizing into a basket linked to the membrane via adaptor proteins is thought to physically pull the membrane inward toward the cytosol to initiate formation of an endocytic vesicle Later the accessory proteins amphiphysin endophilin and dynamin are concentrated at the narrow highly curved membrane necks of deeply invaginated clathrin coated pits where they function in the final scission step that converts the coated pit into a coated vesicle The De Camilli and Hinshaw labs found that these three proteins can each on their own promote the tubulation or vesiculation of membranes reviewed in Hinshaw 2000 This is consistent with amphiphysin endophilin and dynamin directly altering the conformation of the lipid bilayer Various mechanisms for this process have been proposed ranging from oligomerization driven mechanical deformation of the lipid bilayer by dynamin to enzymatic lipid remodeling activities of endophilin that alter the biophysical properties of the lipid bilayer In order for a protein to effect membrane curvature it must first bind to membranes Each of these tubulating proteins binds to membranes often by interacting specifically with the phosphoinositide PtdIns 4 5 P2 PIP2 Recombinant dynamin amphiphysin or endophilin can transform artificial liposomes containing acidic phospholipids and or PIP2 into protein wrapped tubules It has been suggested that dynamin promotes scission through a combination of binding activities GTP driven conformational changes of the dynamin oligomer and additional functions of associated proteins that conspire together to promote fission of the membrane Hinshaw 2000 One concern about the biophysical contortions of the membrane at the constricted neck is the high degree of curvature both positive around the circumference of the neck and negative the sharp bend as viewed perpendicular to the plasma membrane see Figure 1 One model that tried to account for these extremes in curvature suggested that the enzymatic activity of endophilin the condensation of lysophosphatidic acid acyl CoA to form phosphatidic acid altered the curvature of the membrane by physically changing the composition of the phospholipids from inverted cone shaped to cone shaped and thus affected the inherent disposition of the membrane Schmidt et al 1999 However this model is now disfavored since endophilin lacking the enzymatic activity can still tubulate membranes Farsad et al 2001 These final scission events are likely to require quite different changes in membrane structure and curvature as compared to those that initiate bud formation to which we now turn our attention ENTH Domains Studies of epsin by the De Camilli lab Chen et al 1998 and other groups have indicated a role for epsin in endocytosis in particular the highly conserved amino terminal region Wendland et al 1999 which was christened the epsin N terminal homology ENTH domain Kay et al 1998 While many studies have indicated a central role for epsin in the process of endocytosis the precise Cell 144 Figure 1 The Process of Endocytosis Is Depicted as a Series of Events Beginning with the Initiation of a Bud and Culminating in the Release of a Clathrin Coated Vesicle into the Cytoplasm Bud initiation is thought to occur in part by the association of epsin as well as adaptor proteins like AP2 binding sorting signals on receptor tails As the bud forms and constricts the activities of other proteins such as endophilin and amphiphysin cooperate at the edge of the growing clathrin coat to remodel the lipids at the bud neck The GTPase activity of dynamin is required for the scission event that separates the vesicle from the plasma membrane Negative and positive curvature are indicated by arrows in the inset functions of epsin and its ENTH domain