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.

To overcome host defenses, bacterial pathogens of the genus Yersinia inject specific effector proteins into colonized mammalian cells. One such virulence factor, YopJ, inhibits the host inflammatory response and induces apoptosis of immune cells by blocking multiple signaling pathways, including the MAPK and NF-B pathways. In this study, we show that YopJ exerts its deleterious effects by catalyzing the acetylation of two serine residues in the activation loop of the MAP kinase kinase, MEK2. This covalent modification prevents the phosphorylation of these serine residues that is required for activation of MEK2 and downstream signal propagation. We also show that YopJ causes acetylation of a threonine residue in the activation loop of both the ␣ and ␤ subunits of the NF-B pathway kinase, IKK. These results establish a hitherto uncharacterized mode of action for bacterial toxins and suggest the possibility that serine/threonine acetylation may occur even under nonpathogenic conditions and may be a widespread protein modification regulating protein function in eukaryotic cells.inflammation ͉ MEK U nderstanding the mode of action of bacterial toxins has provided insight into the working of mammalian cells especially with regard to signal transduction pathways that impinge upon the activation of the innate immune system (1, 2). Historically, plague has been one of the most devastating diseases to humans, second only to smallpox. The bacillus Yersinia pestis is the causative agent of plague, and two other Yersinia species, Yersinia pseudotuberculosis and Yersinia enterocolitica, cause septicaemic and gastrointestinal disorders (3). These pathogens inject a bouquet of six effector proteins into the mammalian cell cytosol using a type III secretion apparatus (4). These Yersinia outer proteins (Yops) help the pathogen multiply extracellularly in the host by preventing its phagocytosis and by slowing down the onset of the inflammatory response (5). YopE, YopT, and YopO target the Rho family of GTP-binding proteins that control actin cytoskeleton dynamics whereas YopH dephosphorylates focal adhesion proteins, thus inhibiting focal adhesion disassembly. Together, the action of these Yops contributes to the resistance of Yersinia to undergo phagocytosis, a process known to require remodeling of the actin cytoskeleton and of focal adhesions. Suppression of phagocytosis enables Yersinia to evade the macrophage defense network, thereby allowing them to proliferate in Peyer's patches as extracellular microcolonies. The leucine-rich protein, YopM, has been shown to bind to several host cell kinases, resulting in their activation (6). The remaining outer protein, YopJ (also called YopP in Y. enterocolitica) has emerged as an important agent that leads to the reduced host inflammatory response characteristic of Yersinia infections (5). Exposure of macrophages to lipopolysaccharide leads to the activation of NF-B and of several members of the MAPK family that promote the production of proinflammatory cytokines such as TNF-␣. YopJ in...

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.

Translation of aberrant mRNAs can cause ribosomes to stall, leading to collisions with trailing ribosomes. Collided ribosomes are specifically recognized by ZNF598 to initiate protein and mRNA quality control pathways. Here we found using quantitative proteomics of collided ribosomes that EDF1 is a ZNF598-independent sensor of ribosome collisions. EDF1 stabilizes GIGYF2 at collisions to inhibit translation initiation in cis via 4EHP. The GIGYF2 axis acts independently of the ZNF598 axis, but each pathway's output is more pronounced without the other. We propose that the widely conserved and highly abundant EDF1 monitors the transcriptome for excessive ribosome density, then triggers a GIGYF2-mediated response to locally and temporarily reduce ribosome loading. Only when collisions persist is translation abandoned to initiate ZNF598-dependent quality control. This tiered response to ribosome collisions would allow cells to dynamically tune translation rates while ensuring fidelity of the resulting protein products.