Khashayar Farsad

Generation of high curvature membranes mediated by direct endophilin bilayer interactions

Endophilin 1 is a presynaptically enriched protein which binds the GTPase dynamin and the polyphosphoinositide phosphatase synptojanin. Perturbation of endophilin function in cell-free systems and in a living synapse has implicated endophilin in endocytic vesicle budding (Ringstad, N., H. Gad, P. Low, G. Di Paolo, L. Brodin, O. Shupliakov, and P. De Camilli. 1999. Neuron. 24:143–154; Schmidt, A., M. Wolde, C. Thiele, W. Fest, H. Kratzin, A.V. Podtelejnikov, W. Witke, W.B. Huttner, and H.D. Soling. 1999. Nature. 401:133–141; Gad, H., N. Ringstad, P. Low, O. Kjaerulff, J. Gustafsson, M. Wenk, G. Di Paolo, Y. Nemoto, J. Crun, M.H. Ellisman, et al. 2000. Neuron. 27:301–312). Here, we show that purified endophilin can directly bind and evaginate lipid bilayers into narrow tubules similar in diameter to the neck of a clathrin-coated bud, providing new insight into the mechanisms through which endophilin may participate in membrane deformation and vesicle budding. This property of endophilin is independent of its putative lysophosphatydic acid acyl transferase activity, is mediated by its NH2-terminal region, and requires an amino acid stretch homologous to a corresponding region in amphiphysin, a protein previously shown to have similar effects on lipid bilayers (Takei, K., V.I. Slepnev, V. Haucke, and P. De Camilli. 1999. Nat. Cell Biol. 1:33–39). Endophilin cooligomerizes with dynamin rings on lipid tubules and inhibits dynamins GTP-dependent vesiculating activity. Endophilin B, a protein with homology to endophilin 1, partially localizes to the Golgi complex and also deforms lipid bilayers into tubules, underscoring a potential role of endophilin family members in diverse tubulovesicular membrane-trafficking events in the cell.

Generation of Coated Intermediates of Clathrin-Mediated Endocytosis on Protein-Free Liposomes

Clathrin-coated buds and dynamin-coated tubules morphologically similar to corresponding structures observed in synaptic membranes can be generated on protein-free liposomes by incubation with cytosol, or with clathrin coat proteins and purified dynamin, respectively. Dynamin- and clathrin-coated intermediates may form independently of each other, despite the coupling between the two processes typically observed in synaptic membranes. Formation of both structures on liposomes can occur in the absence of nucleotides. These findings indicate that interfaces between lipids and cytosolic proteins are fully sufficient to deform lipids bilayers into buds and tubules. They suggest that a main function of membrane proteins is to act as positive and negative regulators of coat assembly, therefore controlling these processes in time and space.

Mechanisms of membrane deformation.

Membrane traffic requires the generation of high-curvature lipid-bound transport carriers represented by tubules and vesicles. The mechanisms through which membranes are deformed has gained much recent attention. A major advance has been the demonstration that direct interactions between cytosolic proteins and lipid bilayers are important in the acquisition of membrane curvature. Rather than being driven only by the formation of membrane-associated structural scaffolds, membrane deformation requires physical perturbation of the lipid bilayer. A variety of proteins have been identified that directly bind and deform membranes. An emerging theme in this process is the importance of amphipathic peptides that partially penetrate the lipid bilayer.

Dual interaction of synaptotagmin with μ2‐ and α‐adaptin facilitates clathrin‐coated pit nucleation

The synaptic vesicle protein synaptotagmin was proposed to act as a major docking site for the recruitment of clathrin coats implicated in endocytosis, including the recycling of synaptic vesicles. We show here that the C2B domain of synaptotagmin binds μ2‐ and α‐adaptin, two of the four subunits of the endocytic adaptor complex AP‐2. μ2 represents the major interacting subunit of AP‐2 within this complex. Its binding to synaptotagmin is mediated by a site in subdomain B that is distinct from the binding site for tyrosine‐based sorting motifs located in subdomain A. The presence of the C2B domain of synaptotagmin at the surface of liposomes enhances the recruitment of AP‐2 and clathrin. Conversely, perturbation of the interaction between synaptotagmin and AP‐2 by synprint, the cytoplasmic synaptotagmin‐binding domain of N‐type calcium channels, inhibits transferrin internalization in living cells. We conclude that a dual interaction of synaptotagmin with the clathrin adaptor AP‐2 plays a key physiological role in the nucleation of endocytic clathrin‐coated pits.

A putative role for intramolecular regulatory mechanisms in the adaptor function of amphiphysin in endocytosis.

Amphiphysin 1 is a brain-specific protein enriched at the synapse and a major binding partner of several components of the clathrin-mediated endocytic machinery (Proc Natl Acad Sci USA 93 (1996) 331). It interacts with clathrin-coat proteins, dynamin, and membranes (Nat Cell Biol 1 (1999) 33; JBC). A role of amphiphysin in synaptic vesicle recycling is supported by both acute and chronic perturbation studies (Science 276 (1997) 259; Neuron 33 (2002) 789). Here we show that amphiphysin directly stimulates clathrin recruitment onto liposomes in an in vitro assay. Amphiphysin-dependent clathrin-coat recruitment is enhanced by the interaction of amphiphysin with dynamin. We also show that the amphiphysin SH3 domain binds full-length amphiphysin, likely via an internal poly-proline region, and that clathrin recruitment onto liposomes by amphiphysin is enhanced in the presence of the isolated amphiphysin SH3 domain. Expression of a mutant amphiphysin harboring two amino acid substitutions in the SH3 domain, and therefore unable to bind proline-containing motifs, induces an accumulation of large intracellular aggregates including amphiphysin, clathrin, AP-2, and other endocytic proteins, as well as a concomitant block of transferrin endocytosis. Thus, putative intramolecular interactions between the amphiphysin COOH-terminal SH3 domain and its internal poly-proline region may regulate clathrin recruitment onto membranes.