A role for amphiphysin in AP-1/clathrin coat formation

Transport of cargo within the endocytic and secretory pathway is generally mediated by coated vesicles. Clathrin, in combination with different adaptor proteins, is the major coat protein for vesicle formation at the plasma membrane, endosomes, and the trans-Golgi network (TGN). Best characterized is the formation of clathrin coats for endocytosis at the plasma membrane involving the adaptor protein complex AP-2. Clathrin and AP-2 were shown to be at the centre of a complex interactome of proteins accessory to vesicle formation. Considerably less is known about the formation of clathrin coated carriers at the TGN and endosomes, where the adaptor protein complex AP-1 plays a major role. In vitro studies showed the minimal requirements for association of AP-1 to liposomal membranes to be activated ARF1, phosphoinositides, and either sorting signals or unknown cytosolic factors. We have used a liposome floatation assay to identify cytosolic proteins collaborating with AP-1 at the membrane. Separation of proteins from bovine brain cytosol with several chromatographic methods yielded an active fraction containing amphiphysin 1, amphiphysin 2, and endophilin A1. All three proteins are expressed in brain and known to be involved in AP-2/clathrin coat formation. They consist of an N-terminal N-BAR (Bin, amphiphysin, Rvs) domain for dimerization and membrane binding and a C-terminal SH3 (Src homology 3) domain for interaction with dynamin and synaptojanin. Amphiphysin 1 and 2 in addition contain a middle domain with binding sites for adaptors and clathrin. It was proposed that amphiphysins and endophilin are targeted to membranes with high curvature, such as the neck of a forming vesicle, where they recruit dynamin and synaptojanin in preparation for vesicle fission and uncoating. In this thesis, I bacterially expressed and purified all three proteins and tested them in the floatation assay for AP-1 membrane binding activity. Only amphiphysin 2 showed activity, both as a homodimer and as a heterodimer with amphiphysin 1. Activity depended on a motif that was shown to bind to AP-1, AP-2, and clathrin in GST pull-down experiments. Endogenous amphiphysins in primary neurons, as well as transiently expressed in neuronal or fibroblast cell lines, co-localized with AP-1 at the TGN. In addition, when expressed at high levels in neuronal cells, amphiphysins aggregated and interfered dominantly with the TGN localization of AP-1. Both phenomena depended on the presence of the clathrin and adaptor interaction sequence in the amphiphysins. Furthermore, both amphiphysins could be cross-linked to AP-1 in vivo. Our results indicate that amphiphysin 1 and 2 function not only in clathrin coated vesicle formation for endocytosis at the plasma membrane, but are also part of the machinery forming AP-1/clathrin coats at the TGN and endosomes. This suggests that the machineries for CCV formation with AP-1 and AP-2 at different locations in the cell share more components than previously anticipated

Recruitment of coat proteins to liposomes and peptidoliposomes

Intracellular transport within the cell is generally mediated by membrane vesicles. Their formation is typically initiated by activation of small GTPases that then recruit cytosolic proteins to the membrane surface to form a coat, interact with cargo and accessory proteins, and deform the lipid bilayer to produce a transport vesicle. Liposomes proved to be a useful tool to study the molecular mechanisms of these processes in vitro. Here we describe the use of liposomes and peptidoliposomes presenting lipid-coupled cytosolic tails of cargo proteins for the in vitro analysis of the membrane recruitment of AP-1 adaptors in the process of forming AP-1/clathrin coats. AP-1 recruitment is mediated by the GTPase Arf1 and requires specific lipids and cargo signals. Interaction with cargo induces AP-1 oligomerization already in the absence of clathrin. Without cargo peptides, accessory proteins, such as amphiphysin 2, can be identified that stabilize AP-1 binding to liposomal membranes

The BatR/BatS Two-Component Regulatory System Controls the Adaptive Response of Bartonella henselae during Human Endothelial Cell Infection ▿ † ‡

Here, we report the first comprehensive study of Bartonella henselae gene expression during infection of human endothelial cells. Expression of the main cluster of upregulated genes, comprising the VirB type IV secretion system and its secreted protein substrates, is shown to be under the positive control of the transcriptional regulator BatR. We demonstrate binding of BatR to the promoters of the virB operon and a substrate-encoding gene and provide biochemical evidence that BatR and BatS constitute a functional two-component regulatory system. Moreover, in contrast to the acid-inducible (pH 5.5) homologs ChvG/ChvI of Agrobacterium tumefaciens, BatR/BatS are optimally activated at the physiological pH of blood (pH 7.4). By conservation analysis of the BatR regulon, we show that BatR/BatS are uniquely adapted to upregulate a genus-specific virulence regulon during hemotropic infection in mammals. Thus, we propose that BatR/BatS two-component system homologs represent vertically inherited pH sensors that control the expression of horizontally transmitted gene sets critical for the diverse host-associated life styles of the alphaproteobacteria

Interaction of amphiphysins with AP-1 clathrin adaptors at the membrane

The assembly of clathrin/AP-1-coated vesicles on the trans-Golgi network and endosomes is much less studied than of clathrin/AP-2 vesicles at the plasma membrane for endocytosis. In vitro, AP-1 association to protein-free liposomes had been shown to require phosphoinositides, Arf1•GTP, and additional cytosolic factor(s). We have purified an active fraction from brain cytosol and found it to contain amphiphysin 1 and 2 and endophilin A1, three proteins known to be involved in the formation of AP-2/clathrin coats at the plasma membrane. Assays with bacterially expressed and purified proteins showed AP-1 stabilization on liposomes to depend on amphiphysin 2 or the amphiphysin 1/2 heterodimer. Activity is independent of the SH3 domain, but requires the WDLW motif interacting with γ-adaptin. Endogenous amphiphysin in neurons and transfected protein in cell lines colocalize perinuclearly with AP-1 at the trans-Golgi network. This localization depends on the clathrin and adaptor interaction sequence in the amphiphysins and is sensitive to brefeldin A, which inhibits Arf1-dependent AP-1 recruitment. Interaction between AP-1 and amphiphysin 1/2 in vivo was demonstrated by coimmunoprecipitation after crosslinking. These results suggest an involvement of amphiphysins not only with AP-2 at the plasma membrane, but also in AP-1/clathrin coat formation at the trans-Golgi network

Covalently circularized nanodiscs for studying membrane proteins and viral entry

We engineered covalently circularized nanodiscs (cNDs) which, compared with standard nanodiscs, exhibit enhanced stability, defined diameter sizes and tunable shapes. Reconstitution into cNDs enhanced the quality of nuclear magnetic resonance spectra for both VDAC-1, a β-barrel membrane protein, and the G-protein-coupled receptor NTR1, an α-helical membrane protein. In addition, we used cNDs to visualize how simple, nonenveloped viruses translocate their genomes across membranes to initiate infection