SummaryThe AP2 adaptor complex (α, β2, σ2, and μ2 subunits) crosslinks the endocytic clathrin scaffold to PtdIns4,5P2-containing membranes and transmembrane protein cargo. In the “locked” cytosolic form, AP2's binding sites for the two endocytic motifs, YxxΦ on the C-terminal domain of μ2 (C-μ2) and [ED]xxxL[LI] on σ2, are blocked by parts of β2. Using protein crystallography, we show that AP2 undergoes a large conformational change in which C-μ2 relocates to an orthogonal face of the complex, simultaneously unblocking both cargo-binding sites; the previously unstructured μ2 linker becomes helical and binds back onto the complex. This structural rearrangement results in AP2's four PtdIns4,5P2- and two endocytic motif-binding sites becoming coplanar, facilitating their simultaneous interaction with PtdIns4,5P2/cargo-containing membranes. Using a range of biophysical techniques, we show that the endocytic cargo binding of AP2 is driven by its interaction with PtdIns4,5P2-containing membranes.

SummaryMost transmembrane proteins are selected as transport vesicle cargo through the recognition of short, linear amino acid motifs in their cytoplasmic portions by vesicle coat proteins. In the case of clathrin-coated vesicles (CCVs) the motifs are recognised by clathrin adaptors. The AP2 adaptor complex (subunits α,β2,μ2,σ2) recognises both major endocytic motifs: YxxΦ motifs 1 and [DE]xxxL[LI] acidic dileucine motifs. Here we describe the binding of AP2 to the endocytic dileucine motif from CD4 2. The major recognition events are the two leucine residues binding in hydrophobic pockets on σ2. The hydrophilic residue four residues upstream from the first leucine sits on a positively charged patch made from residues on σ2 and α subunits. Mutations in key residues inhibit the binding of AP2 to ‘acidic dileucine’ motifs displayed in liposomes containing PtdIns4,5P2, but do not affect binding to YxxΦ motifs via μ2. In the ‘inactive’ AP2 core structure 3, both motif binding sites are blocked by different parts of the β2 subunit. To allow a dileucine motif to bind, the β2 N-terminus is displaced and becomes disordered; however, in this structure the YxxΦ binding site on μ2 remains blocked.

Clathrin-mediated endocytosis (CME) is vital for the internalization of most cell-surface proteins. In CME, plasma membrane-binding clathrin adaptors recruit and polymerize clathrin to form clathrin-coated 'pits' into which cargo is sorted. AP2 is the most abundant adaptor, and is pivotal to CME. By determining a new structure of AP2 that includes the clathrin-binding β2-hinge and developing an AP2-dependent budding assay, we reveal the existence of an autoinhibitory mechanism that prevents clathrin recruitment by cytosolic AP2. A large-scale conformational change driven by the plasma membrane phosphoinositide PtdIns(4,5)P 2 and cargo relieves this autoinhibition, so triggering clathrin recruitment and hence clathrin-coated bud formation. This molecular switching mechanism constitutes an unsuspected layer of regulation that couples AP2's membrane recruitment to its key functions of cargo and clathrin binding.Clathrin adaptors provide an essential physical bridge connecting clathrin, which itself lacks membrane binding activity (1), to the membrane and to embedded transmembrane protein cargo. A central player in CME is the AP2 (Assembly Polypeptide 2) complex, (Figs 1A, S1), which both coordinates CCP formation and binds the many cargo proteins that contain 'acidic dileucine' and Yxxφ endocytic motifs (φ denotes a bulky hydrophobic residue) through its membrane proximal core (2, 3). Cargo binding is activated by a large-scale conformational change from the 'locked' or 'inactive' cytosolic form to an 'open' or 'active' form driven by localization to membranes containing the plasma membrane phosphoinositide PtdIns(4,5)P 2 (4, 5). The C-terminal 'appendages' of the α and β2 subunits bind other clathrin adaptors as well as CCV (clathrin-coated vesicle) assembly and disassembly accessory factors (3,(6)(7)(8) Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts from the β2-trunk binds the N-terminal beta-propeller of the clathrin heavy chain using a canonical clathrin box motif (LLNLD; Fig 1A,B (9)). The β2 appendage domain also binds clathrin, albeit weakly, but both interactions are necessary for robust clathrin binding (10).A version of AP2 comprising full-length β2, μ2 and σ2 subunits, and the α-trunk domain, (FLβ.AP2) (Fig 1B)(11) was expressed in E.coli, avoiding contamination with other CCV components inherent to purification from brain tissue (12, 13). Despite most FLβ.AP2 possessing an intact β2 subunit (Fig 1C-E), it bound clathrin very poorly in pulldowns when immobilized on either glutathione sepharose beads ( Fig 1C) or via its N-terminal His6 tag (similarly positioned to the β2 PtdIns(4,5)P 2 binding site Fig1B (4, 5).) to liposomes containing the nickel-attached lipid NiNTA-DGS ( Fig 1E): in both cases the FLβ.AP2 will be in its locked cytosolic conformation (4). FLβ.AP2 also failed to stimulate clathrin cage assembly efficiently at physiological pH ( Fig 1D). In contrast, the isolated β2 hingeappendage ('GST-β2-h+app', Fig S1) bound clathrin efficiently ( Fig 1C) and stimu...

SummaryThe size of endocytic clathrin-coated vesicles (CCVs) is remarkably uniform, suggesting that it is optimized to achieve the appropriate levels of cargo and lipid internalization. The three most abundant proteins in mammalian endocytic CCVs are clathrin and the two cargo-selecting, clathrin adaptors, CALM and AP2. Here we demonstrate that depletion of CALM causes a substantial increase in the ratio of “open” clathrin-coated pits (CCPs) to “necked”/“closed” CCVs and a doubling of CCP/CCV diameter, whereas AP2 depletion has opposite effects. Depletion of either adaptor, however, significantly inhibits endocytosis of transferrin and epidermal growth factor. The phenotypic effects of CALM depletion can be rescued by re-expression of wild-type CALM, but not with CALM that lacks a functional N-terminal, membrane-inserting, curvature-sensing/driving amphipathic helix, the existence and properties of which are demonstrated. CALM is thus a major factor in controlling CCV size and maturation and hence in determining the rates of endocytic cargo uptake.

We describe a completely in vitro high-throughput screening system for directed evolution of enzymes based on in vitro compartmentalization (IVC). Single genes are transcribed and translated inside the aqueous droplets of a water-in-oil emulsion. Enzyme activity generates a fluorescent product and, after conversion into a water-in-oil-in-water double emulsion, fluorescent droplets are sorted using a fluorescence-activated cell sorter (FACS). Earlier in vivo studies have demonstrated that Ebg, a protein of unknown function, can evolve to allow Escherichia coli lacking the lacZ beta-galactosidase gene to grow on lactose. Here we demonstrate that we can evolve Ebg into an enzyme with significant beta-galactosidase activity in vitro. Only two specific mutations were ever seen to provide this improvement in Ebg beta-galactosidase activity in vivo. In contrast, nearly all the improved beta-galactosidases selected in vitro resulted from different mutations.

No abstract

By compartmentalizing reactions in aqueous microdroplets of water-in-oil emulsions, reaction volumes can be reduced by factors of up to 10(9) compared to conventional microtitre-plate based systems. This allows massively parallel processing of as many as 10(10) reactions in a total volume of only 1 ml of emulsion. This review describes the use of emulsions for directed evolution of proteins and RNAs, and for performing polymerase chain reactions (PCRs). To illustrate these applications we describe certain specific experiments, each of which exemplifies a different facet of the technique, in some detail. These examples include directed evolution of Diels-Alderase and RNA ligase ribozymes and several classes of protein enzymes, including DNA polymerases, phosphotriesterases, beta-galactosidases and thiolactonases. We also describe the application of emulsion PCR to screen for rare mutations and for new ultra-high throughput sequencing technologies. Finally, we discuss the recent development of microfluidic tools for making and manipulating microdroplets and their likely impact on the future development of the field.

In vitro compartmentalization (IVC) has previously been used to evolve protein enzymes. Here, we demonstrate how IVC can be applied to select RNA enzymes (ribozymes) for a property that has previously been unselectable: true intermolecular catalysis. Libraries containing 10 11 ribozyme genes are compartmentalized in the aqueous droplets of a water-in-oil emulsion, such that most droplets contain no more than one gene, and transcribed in situ. By coencapsulating the gene, RNA, and the substrates͞products of the catalyzed reaction, ribozymes can be selected for all enzymatic properties: substrate recognition, product formation, rate acceleration, and turnover. Here we exploit the complementarity of IVC with systematic evolution of ligands by exponential enrichment (SELEX), which allows selection of larger libraries (>10 15 ) and for very small rate accelerations (kcat͞kuncat) but only selects for intramolecular single-turnover reactions. We selected Ϸ10 14 random RNAs for Diels-Alderase activity with five rounds of SELEX, then six to nine rounds with IVC. All selected ribozymes catalyzed the Diels-Alder reaction in a truly bimolecular fashion and with multiple turnover. Nearly all ribozymes selected by using eleven rounds of SELEX alone contain a common catalytic motif. Selecting with SELEX then IVC gave ribozymes with significant sequence variations in this catalytic motif and ribozymes with completely novel motifs. Interestingly, the catalytic properties of all of the selected ribozymes were quite similar. The ribozymes are strongly product inhibited, consistent with the Diels-Alder transition state closely resembling the product. More efficient Diels-Alderases may need to catalyze a second reaction that transforms the product and prevents product inhibition.emulsion ͉ RNA ͉ intermolecular catalysis T he Diels-Alder [4 ϩ 2] cycloaddition reaction (1) between a 1,3 diene and an alkene dienophile is one of the most useful and important in synthetic chemistry, because it allows the formation of six-membered rings by making two simultaneous C-C bonds and at the same time generates up to four chiral centers (2). However, nature rarely seems to use this mechanism for C-C bond formation and instead prefers other mechanisms such as the aldol condensation reaction. Although Ͼ100 natural products have been proposed to be Diels-Alder cycloadducts (3), direct evidence from biosynthetic studies with purified or partially purified enzymes is available for only three naturally occurring Diels-Alder reactions (4).However, several antibodies and RNAs that catalyze Diels-Alder reactions have been generated in the laboratory. The DielsAlderase antibodies were raised against a hapten that mimicked either the Diels-Alder adduct or the transition state of the desired reactants (reviewed in ref. 5). Diels-Alderase ribozymes with both pyridyl-modified (6, 7) and unmodified RNA (8) were generated by using a variation on systematic evolution of ligands by exponential enrichment (SELEX) (9, 10). One substrate is physically tethered to the...