Cargo and Dynamin Regulate Clathrin-Coated Pit Maturation

Total internal reflection fluorescence microscopy (TIR-FM) has become a powerful tool for studying clathrin-mediated endocytosis. However, due to difficulties in tracking and quantifying their heterogeneous dynamic behavior, detailed analyses have been restricted to a limited number of selected clathrin-coated pits (CCPs). To identify intermediates in the formation of clathrin-coated vesicles and factors that regulate progression through these stages, we used particle-tracking software and statistical methods to establish an unbiased and complete inventory of all visible CCP trajectories. We identified three dynamically distinct CCP subpopulations: two short-lived subpopulations corresponding to aborted intermediates, and one longer-lived productive subpopulation. In a manner dependent on AP2 adaptor complexes, increasing cargo concentration significantly enhances the maturation efficiency of productive CCPs, but has only minor effects on their lifetimes. In contrast, small interfering RNA (siRNA) depletion of dynamin-2 GTPase and reintroduction of wild-type or mutant dynamin-1 revealed dynamin's role in controlling the turnover of abortive intermediates and the rate of CCP maturation. From these data, we infer the existence of an endocytic restriction or checkpoint, responsive to cargo and regulated by dynamin

Protein flexibility is required for vesicle tethering at the Golgi

Abstract The Golgi is decorated with coiled-coil proteins that may extend long distances to help vesicles find their targets. GCC185 is a trans Golgi-associated protein that captures vesicles inbound from late endosomes. Although predicted to be relatively rigid and highly extended, we show that flexibility in a central region is required for GCC185's ability to function in a vesicle tethering cycle. Proximity ligation experiments show that that GCC185's N-and C-termini are within <40 nm of each other on the Golgi. In physiological buffers without fixatives, atomic force microscopy reveals that GCC185 is shorter than predicted, and its flexibility is due to a central bubble that represents local unwinding of specific sequences. Moreover, 85% of the N-termini are splayed, and the splayed N-terminus can capture transport vesicles in vitro. These unexpected features support a model in which GCC185 collapses onto the Golgi surface, perhaps by binding to Rab GTPases, to mediate vesicle tethering

Insights into Mad2 Regulation in the Spindle Checkpoint Revealed by the Crystal Structure of the Symmetric Mad2 Dimer

In response to misaligned sister chromatids during mitosis, the spindle checkpoint protein Mad2 inhibits the anaphase-promoting complex or cyclosome (APC/C) through binding to its mitotic activator Cdc20, thus delaying anaphase onset. Mad1, an upstream regulator of Mad2, forms a tight core complex with Mad2 and facilitates Mad2 binding to Cdc20. In the absence of its binding proteins, free Mad2 has two natively folded conformers, termed N1-Mad2/open-Mad2 (O-Mad2) and N2-Mad2/closed Mad2 (C-Mad2), with C-Mad2 being more active in APC/CCdc20 inhibition. Here, we show that whereas O-Mad2 is monomeric, C-Mad2 forms either symmetric C-Mad2–C-Mad2 (C–C) or asymmetric O-Mad2–C-Mad2 (O–C) dimers. We also report the crystal structure of the symmetric C–C Mad2 dimer, revealing the basis for the ability of unliganded C-Mad2, but not O-Mad2 or liganded C-Mad2, to form symmetric dimers. A Mad2 mutant that predominantly forms the C–C dimer is functional in vitro and in living cells. Finally, the Mad1–Mad2 core complex facilitates the conversion of O-Mad2 to C-Mad2 in vitro. Collectively, our results establish the existence of a symmetric Mad2 dimer and provide insights into Mad1-assisted conformational activation of Mad2 in the spindle checkpoint

Structural Basis for a Munc13–1 Homodimer to Munc13–1/RIM Heterodimer Switch

C (2) domains are well characterized as Ca (2+)/phospholipid-binding modules, but little is known about how they mediate protein–protein interactions. In neurons, a Munc13–1 C (2)A-domain/RIM zinc-finger domain (ZF) heterodimer couples synaptic vesicle priming to presynaptic plasticity. We now show that the Munc13–1 C (2)A domain homodimerizes, and that homodimerization competes with Munc13–1/RIM heterodimerization. X-ray diffraction studies guided by nuclear magnetic resonance (NMR) experiments reveal the crystal structures of the Munc13–1 C (2)A-domain homodimer and the Munc13–1 C (2)A-domain/RIM ZF heterodimer at 1.44 Å and 1.78 Å resolution, respectively. The C (2)A domain adopts a β-sandwich structure with a four-stranded concave side that mediates homodimerization, leading to the formation of an eight-stranded β-barrel. In contrast, heterodimerization involves the bottom tip of the C (2)A-domain β-sandwich and a C-terminal α-helical extension, which wrap around the RIM ZF domain. Our results describe the structural basis for a Munc13–1 homodimer–Munc13–1/RIM heterodimer switch that may be crucial for vesicle priming and presynaptic plasticity, uncovering at the same time an unexpected versatility of C (2) domains as protein–protein interaction modules, and illustrating the power of combining NMR spectroscopy and X-ray crystallography to study protein complexes

Munc18-Bound Syntaxin Readily Forms SNARE Complexes with Synaptobrevin in Native Plasma Membranes

Munc18–1, a protein essential for regulated exocytosis in neurons and neuroendocrine cells, belongs to the family of Sec1/Munc18-like (SM) proteins. In vitro, Munc18–1 forms a tight complex with the SNARE syntaxin 1, in which syntaxin is stabilized in a closed conformation. Since closed syntaxin is unable to interact with its partner SNAREs SNAP-25 and synaptobrevin as required for membrane fusion, it has hitherto not been possible to reconcile binding of Munc18–1 to syntaxin 1 with its biological function. We now show that in intact and exocytosis-competent lawns of plasma membrane, Munc18–1 forms a complex with syntaxin that allows formation of SNARE complexes. Munc18–1 associated with membrane-bound syntaxin 1 can be effectively displaced by adding recombinant synaptobrevin but not syntaxin 1 or SNAP-25. Displacement requires the presence of endogenous SNAP-25 since no displacement is observed when chromaffin cell membranes from SNAP-25–deficient mice are used. We conclude that Munc18–1 allows for the formation of a complex between syntaxin and SNAP-25 that serves as an acceptor for vesicle-bound synaptobrevin and that thus represents an intermediate in the pathway towards exocytosis

Role of the AP2 β-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 Liganding of Glycolipid Transfer Protein Is Controlled by Glycolipid Acyl Structure

Glycosphingolipids (GSLs) play major roles in cellular growth and development. Mammalian glycolipid transfer proteins (GLTPs) are potential regulators of cell processes mediated by GSLs and display a unique architecture among lipid binding/transfer proteins. The GLTP fold represents a novel membrane targeting/interaction domain among peripheral proteins. Here we report crystal structures of human GLTP bound to GSLs of diverse acyl chain length, unsaturation, and sugar composition. Structural comparisons show a highly conserved anchoring of galactosyl- and lactosyl-amide headgroups by the GLTP recognition center. By contrast, acyl chain chemical structure and occupancy of the hydrophobic tunnel dictate partitioning between sphingosine-in and newly-observed sphingosine-out ligand-binding modes. The structural insights, combined with computed interaction propensity distributions, suggest a concerted sequence of events mediated by GLTP conformational changes during GSL transfer to and/or from membranes, as well as during GSL presentation and/or transfer to other proteins

Modification of a Hydrophobic Layer by a Point Mutation in Syntaxin 1A Regulates the Rate of Synaptic Vesicle Fusion

Both constitutive secretion and Ca(2+)-regulated exocytosis require the assembly of the soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complexes. At present, little is known about how the SNARE complexes mediating these two distinct pathways differ in structure. Using the Drosophila neuromuscular synapse as a model, we show that a mutation modifying a hydrophobic layer in syntaxin 1A regulates the rate of vesicle fusion. Syntaxin 1A molecules share a highly conserved threonine in the C-terminal +7 layer near the transmembrane domain. Mutation of this threonine to isoleucine results in a structural change that more closely resembles those found in syntaxins ascribed to the constitutive secretory pathway. Flies carrying the I254 mutant protein have increased levels of SNARE complexes and dramatically enhanced rate of both constitutive and evoked vesicle fusion. In contrast, overexpression of the T254 wild-type protein in neurons reduces vesicle fusion only in the I254 mutant background. These results are consistent with molecular dynamics simulations of the SNARE core complex, suggesting that T254 serves as an internal brake to dampen SNARE zippering and impede vesicle fusion, whereas I254 favors fusion by enhancing intermolecular interaction within the SNARE core complex

A Promiscuous DNA Packaging Machine from Bacteriophage T4

Packaged viral genome can be removed from bacteriophage T4 capsid and the capsid refilled with any double-stranded DNA, single or multiple molecules, using a powerful ATP-fueled DNA packaging machine

The Mechanochemistry of Endocytosis

An integrated theoretical model reveals how the chemical and the mechanical aspects of endocytosis are coordinated coherently in yeast cells, driving progression through the endocytic pathway and ensuring efficient vesicle scission in vivo