Marijn G. J. Ford

Curvature of clathrin-coated pits driven by epsin

Clathrin-mediated endocytosis involves cargo selection and membrane budding into vesicles with the aid of a protein coat. Formation of invaginated pits on the plasma membrane and subsequent budding of vesicles is an energetically demanding process that involves the cooperation of clathrin with many different proteins. Here we investigate the role of the brain-enriched protein epsin 1 in this process. Epsin is targeted to areas of endocytosis by binding the membrane lipid phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2). We show here that epsin 1 directly modifies membrane curvature on binding to PtdIns(4,5)P2 in conjunction with clathrin polymerization. We have discovered that formation of an amphipathic α-helix in epsin is coupled to PtdIns(4,5)P2 binding. Mutation of residues on the hydrophobic region of this helix abolishes the ability to curve membranes. We propose that this helix is inserted into one leaflet of the lipid bilayer, inducing curvature. On lipid monolayers epsin alone is sufficient to facilitate the formation of clathrin-coated invaginations.

Role of the Ap2 Beta-Appendage Hub in Recruiting Partners for Clathrin-Coated Vesicle Assembly.

Adaptor protein complex 2 α and β-appendage domains act as hubs for the assembly of accessory protein networks involved in clathrin-coated vesicle formation. We identify a large repertoire of β-appendage interactors by mass spectrometry. These interact with two distinct ligand interaction sites on the β-appendage (the “top” and “side” sites) that bind motifs distinct from those previously identified on the α-appendage. We solved the structure of the β-appendage with a peptide from the accessory protein Eps15 bound to the side site and with a peptide from the accessory cargo adaptor β-arrestin bound to the top site. We show that accessory proteins can bind simultaneously to multiple appendages, allowing these to cooperate in enhancing ligand avidities that appear to be irreversible in vitro. We now propose that clathrin, which interacts with the β-appendage, achieves ligand displacement in vivo by self-polymerisation as the coated pit matures. This changes the interaction environment from liquid-phase, affinity-driven interactions, to interactions driven by solid-phase stability (“matricity”). Accessory proteins that interact solely with the appendages are thereby displaced to areas of the coated pit where clathrin has not yet polymerised. However, proteins such as β-arrestin (non-visual arrestin) and autosomal recessive hypercholesterolemia protein, which have direct clathrin interactions, will remain in the coated pits with their interacting receptors.

The crystal structure of dynamin

Dynamin-related proteins (DRPs) are multi-domain GTPases that function via oligomerization and GTP-dependent conformational changes to play central roles in regulating membrane structure across phylogenetic kingdoms. How DRPs harness self-assembly and GTP-dependent conformational changes to remodel membranes is not understood. Here we present the crystal structure of an assembly-deficient mammalian endocytic DRP, dynamin 1, lacking the proline-rich domain, in its nucleotide-free state. The dynamin 1 monomer is an extended structure with the GTPase domain and bundle signalling element positioned on top of a long helical stalk with the pleckstrin homology domain flexibly attached on its opposing end. Dynamin 1 dimer and higher order dimer multimers form via interfaces located in the stalk. Analysis of these interfaces provides insight into DRP family member specificity and regulation and provides a framework for understanding the biogenesis of higher order DRP structures and the mechanism of DRP-mediated membrane scission events.

Evolving nature of the AP2 α-appendage hub during clathrin-coated vesicle endocytosis

Clathrin‐mediated endocytosis involves the assembly of a network of proteins that select cargo, modify membrane shape and drive invagination, vesicle scission and uncoating. This network is initially assembled around adaptor protein (AP) appendage domains, which are protein interaction hubs. Using crystallography, we show that FxDxF and WVxF peptide motifs from synaptojanin bind to distinct subdomains on α‐appendages, called ‘top’ and side’ sites. Appendages use both these sites to interact with their binding partners in vitro and in vivo. Occupation of both sites simultaneously results in high‐affinity reversible interactions with lone appendages (e.g. eps15 and epsin1). Proteins with multiple copies of only one type of motif bind multiple appendages and so will aid adaptor clustering. These clustered α(appendage)‐hubs have altered properties where they can sample many different binding partners, which in turn can interact with each other and indirectly with clathrin. In the final coated vesicle, most appendage binding partners are absent and thus the functional status of the appendage domain as an interaction hub is temporal and transitory giving directionality to vesicle assembly.

Membrane fission by dynamin: what we know and what we need to know.

The large GTPase dynamin is the first protein shown to catalyze membrane fission. Dynamin and its related proteins are essential to many cell functions, from endocytosis to organelle division and fusion, and it plays a critical role in many physiological functions such as synaptic transmission and muscle contraction. Research of the past three decades has focused on understanding how dynamin works. In this review, we present the basis for an emerging consensus on how dynamin functions. Three properties of dynamin are strongly supported by experimental data: first, dynamin oligomerizes into a helical polymer; second, dynamin oligomer constricts in the presence of GTP; and third, dynamin catalyzes membrane fission upon GTP hydrolysis. We present the two current models for fission, essentially diverging in how GTP energy is spent. We further discuss how future research might solve the remaining open questions presently under discussion.

The Conserved Isoleucine–Valine–Phenylalanine Motif Couples Activation State and Endocytic Functions of β‐Arrestins

Beta‐arrestins (βarrs) play a central role in the regulation of G‐protein‐coupled receptors (GPCRs). Their binding to phosphorylated activated GPCRs induces a conformational transition to an active state resulting in the release of their flexible C‐terminal tail. Binding sites for clathrin and the adaptor protein (AP)‐2 clathrin adaptor complex are then unmasked, which drive the recruitment of βarrs‐GPCR complexes into clathrin‐coated pits (CCPs). A conserved isoleucine‐valine‐phenylalanine (IVF) motif of the C‐terminal tail controls βarr activation through intramolecular interactions. Here, we provide structural, biochemical and functional evidence in living cells that the IVF motif also controls binding to AP‐2. While the F residue is directly involved in AP‐2 binding, substitutions of I and V residues, markedly enhanced affinity for AP‐2 resulting in active βarr mutants, which are constitutively targeted to CCPs in the absence of any GPCR activation. Conformational change and endocytic functions of βarrs thus appear to be coordinated via the complex molecular interactions established by the IVF motif.

Solitary and repetitive binding motifs for the AP2 complex alpha-appendage in amphiphysin and other accessory proteins

Adaptor protein (AP) complexes bind to transmembrane proteins destined for internalization and to membrane lipids, so linking cargo to the accessory internalization machinery. This machinery interacts with the appendage domains of APs, which have platform and β-sandwich subdomains, forming the binding surfaces for interacting proteins. Proteins that interact with the subdomains do so via short motifs, usually found in regions of low structural complexity of the interacting proteins. So far, up to four motifs have been identified that bind to and partially compete for at least two sites on each of the appendage domains of the AP2 complex. Motifs in individual accessory proteins, their sequential arrangement into motif domains, and partial competition for binding sites on the appendage domains coordinate the formation of endocytic complexes in a temporal and spatial manner. In this work, we examine the dominant interaction sequence in amphiphysin, a synapse-enriched accessory protein, which generates membrane curvature and recruits the scission protein dynamin to the necks of coated pits, for the platform subdomain of the α-appendage. The motif domain of amphiphysin1 contains one copy of each of a DX(F/W) and FXDXF motif. We find that the FXDXF motif is the main determinant for the high affinity interaction with the α-adaptin appendage. We describe the optimal sequence of the FXDXF motif using thermodynamic and structural data and show how sequence variation controls the affinities of these motifs for the α-appendage.

Pib2 and the EGO complex are both required for activation of TORC1

ABSTRACT The TORC1 complex is a key regulator of cell growth and metabolism in Saccharomyces cerevisiae. The vacuole-associated EGO complex couples activation of TORC1 to the availability of amino acids, specifically glutamine and leucine. The EGO complex is also essential for reactivation of TORC1 following rapamycin-induced growth arrest and for its distribution on the vacuolar membrane. Pib2, a FYVE-containing phosphatidylinositol 3-phosphate (PI3P)-binding protein, is a newly discovered and poorly characterized activator of TORC1. Here, we show that Pib2 is required for reactivation of TORC1 following rapamycin-induced growth arrest. Pib2 is required for EGO complex-mediated activation of TORC1 by glutamine and leucine as well as for redistribution of Tor1 on the vacuolar membrane. Therefore, Pib2 and the EGO complex cooperate to activate TORC1 and connect phosphoinositide 3-kinase (PI3K) signaling and TORC1 activity. Summary: Pib2 and the EGO complex function in the same molecular pathway that leads to the activation of TORC1, a key regulator of cell growth.

Ivy1 is a negative regulator of Gtr-dependent TORC1 activation

ABSTRACT The highly conserved TORC1 complex controls cell growth in response to nutrients, especially amino acids. The EGO complex activates TORC1 in response to glutamine and leucine. Here, we demonstrate that the I-BAR domain-containing protein Ivy1 colocalizes with Gtr1 and Gtr2, a heterodimer of small GTPases that are part of the EGO complex. Ivy1 is a negative regulator of Gtr-induced TORC1 activation, and is contained within puncta associated with the vacuolar membrane in cells grown in nutrient-rich medium or after brief nitrogen starvation. Addition of glutamine to nitrogen-starved cells leads to dissipation of Ivy1 puncta and redistribution of Ivy1 throughout the vacuolar membrane. Continued stimulation with glutamine results in concentration of Ivy1 within vacuolar membrane invaginations and its spatial separation from the EGO complex components Gtr1 and Gtr2. Disruption of vacuolar membrane invagination is associated with persistent mislocalization of Ivy1 across the vacuolar membrane and inhibition of TORC1 activity. Together, our findings illustrate a novel negative-feedback pathway that is exerted by Ivy1 on Gtr-dependent TORC1 signaling and provide insight into a potential molecular mechanism underlying TORC1 activation by vacuolar membrane remodeling. Summary: The I-BAR domain-containing protein Ivy1 colocalizes with the EGO complex components Gtr1 and Gtr2 and regulates their activation.