Stefan Höning

A Large Scale Conformational Change Couples Membrane Recruitment to Cargo Binding in the Ap2 Clathrin Adaptor Complex

Summary The 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, AP2s 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 AP2s 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.

A structural explanation for the binding of endocytic dileucine motifs by the AP2 complex

Most 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. For clathrin-coated vesicles, the motifs are recognized by clathrin adaptors. The AP2 adaptor complex (subunits α, β2, μ2 and σ2) recognizes both major endocytic motifs: YxxΦ motifs (where Φ can be F, I, L, M or V) and [ED]xxxL[LI] acidic dileucine motifs. Here we describe the binding of AP2 to the endocytic dileucine motif from CD4 (ref. 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 the σ2 and α subunits. Mutations in key residues inhibit the binding of AP2 to ‘acidic dileucine’ motifs displayed in liposomes containing phosphatidylinositol-4,5-bisphosphate, but do not affect binding to YxxΦ motifs through μ2. In the ‘inactive’ AP2 core structure both motif-binding sites are blocked by different parts of the β2 subunit. To allow a dileucine motif to bind, the β2 amino terminus is displaced and becomes disordered; however, in this structure the YxxΦ-binding site on μ2 remains blocked.

The Molecular Basis for the Endocytosis of Small R-SNAREs by the Clathrin Adaptor CALM

SNAREs provide a large part of the specificity and energy needed for membrane fusion and, to do so, must be localized to their correct membranes. Here, we show that the R-SNAREs VAMP8, VAMP3, and VAMP2, which cycle between the plasma membrane and endosomes, bind directly to the ubiquitously expressed, PtdIns4,5P(2)-binding, endocytic clathrin adaptor CALM/PICALM. X-ray crystallography shows that the N-terminal halves of their SNARE motifs bind the CALM(ANTH) domain as helices in a manner that mimics SNARE complex formation. Mutation of residues in the CALM:SNARE interface inhibits binding in vitro and prevents R-SNARE endocytosis in vivo. Thus, CALM:R-SNARE interactions ensure that R-SNAREs, required for the fusion of endocytic clathrin-coated vesicles with endosomes and also for subsequent postendosomal trafficking, are sorted into endocytic vesicles. CALMs role in directing the endocytosis of small R-SNAREs may provide insight into the association of CALM/PICALM mutations with growth retardation, cognitive defects, and Alzheimers disease.

Hypoxia-inducible protein 2 is a novel lipid droplet protein and a specific target gene of hypoxia-inducible factor-1

Hypoxia‐inducible protein 2 (HIG2) has been implicated in canonical Wnt signaling, both as target and activator. The potential link between hypoxia and an oncogenic signaling pathway might play a pivotal role in renal clear‐cell carcinoma characterized by constitutive activation of hypoxia‐inducible factors (HIFs), and hence prompted us to analyze HIG2 regulation and function in detail. HIG2 was up‐regulated by hypoxia and HIF inducers in all cell types and mouse organs investigated and abundantly expressed in renal clear‐cell carcinomas. Promoter analyses, gel shifts, and siRNA studies revealed that HIG2 is a direct and specific target of HIF‐1, but not responsive to HIF‐2. Surprisingly, HIG2 was not secreted, and HIG2 overexpression neither stimulated proliferation nor activated Wnt signaling. Instead, we show that HIG2 decorates the hemimembrane of lipid droplets, whose number and size increase on hypoxic inhibition of fatty acid β‐oxidation, and colocalizes with the lipid droplet proteins adipophilin and TIP47. Normoxic overexpression of HIG2 was sufficient to increase neutral lipid deposition in HeLa cells and stimulated cytokine expression. HIG2 could be detected in atherosclerotic arteries and fatty liver disease, suggesting that this ubiquitously inducible HIF‐1 target gene may play an important functional role in diseases associated with pathological lipid accumulation.—Gimm, T., Wiese, M., Teschemacher, B., Deggerich, A., Schödel, J., Knaup, K. X., Hackenbeck, T., Hellerbrand, C., Amann, K., Wiesener, M. S., Honing, S., Eckardt, K.‐U., Warnecke, C. Hypoxia‐inducible protein 2 is a novel lipid droplet protein and a specific target gene of hypoxia‐inducible factor‐1. FASEB J. 24, 4443–4458 (2010). www.fasebj.org

AP2 controls clathrin polymerization with a membrane-activated switch

A membrane-activated switch to bind clathrin Clathrin-mediated endocytosis—the process by which cells take up nutrients and signals within clathrin-coated vesicles—is very well understood. Kelly et al. reveal an unanticipated layer of regulation in this process. The proteins AP2 and clathrin are the major constituents of endocytic clathrin-coated vesicles. AP2 and clathrin stick together through a clathrin-binding motif in AP2. The authors now show that AP2s clathrin-binding motif is normally buried within the core of the AP2 protein. AP2 only ejects its clathrin-binding motif and recruits clathrin if it is associated with the correct cell membrane and an endocytic cargo. Science, this issue p. 459 An autoinhibitory mechanism prevents clathrin recruitment by cytosolic AP2. 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. Assembly polypeptide 2 (AP2) is the most abundant adaptor and is pivotal to CME. Here, we determined a structure of AP2 that includes the clathrin-binding β2 hinge and developed an AP2-dependent budding assay. Our findings suggest that an autoinhibitory mechanism prevents clathrin recruitment by cytosolic AP2. A large-scale conformational change driven by the plasma membrane phosphoinositide phosphatidylinositol 4,5-bisphosphate and cargo relieves this autoinhibition, triggering clathrin recruitment and hence clathrin-coated bud formation. This molecular switching mechanism can couple AP2’s membrane recruitment to its key functions of cargo and clathrin binding.

CALM Regulates Clathrin-Coated Vesicle Size and Maturation by Directly Sensing and Driving Membrane Curvature

Summary The 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.

Structural requirements for PACSIN/syndapin operation during zebrafish embryonic notochord development

PACSIN/Syndapin proteins are membrane-active scaffolds that participate in endocytosis. The structure of the Drosophila Syndapin N-terminal EFC domain reveals a crescent shaped antiparallel dimer with a high affinity for phosphoinositides and a unique membrane-inserting prong upon the concave surface. Combined structural, biochemical and reverse genetic approaches in zebrafish define an important role for Syndapin orthologue, Pacsin3, in the early formation of the notochord during embryonic development. In pacsin3-morphant embryos, midline convergence of notochord precursors is defective as axial mesodermal cells fail to polarize, migrate and differentiate properly. The pacsin3 morphant phenotype of a stunted body axis and contorted trunk is rescued by ectopic expression of Drosophila Syndapin, and depends critically on both the prong that protrudes from the surface of the bowed Syndapin EFC domain and the ability of the antiparallel dimer to bind tightly to phosphoinositides. Our data confirm linkage between directional migration, endocytosis and cell specification during embryonic morphogenesis and highlight a key role for Pacsin3 in this coupling in the notochord.

Polarized transport of Alzheimer amyloid precursor protein is mediated by adaptor protein complex AP1-1B.

Alzheimer amyloid precursor protein (APP) is the precursor for the Aβ peptide involved in pathogenesis of Alzheimers disease. The soluble ectodomain fragment of APP (sAPP) functions as a growth factor for epithelial cells, suggesting an important function for APP outside neuronal tissue. Previous studies have shown that in polarized epithelial cells, APP is targeted to the basolateral domain. Tyr653 within the cytoplasmic tail of APP mediates the basolateral targeting of APP, but the sorting machinery that binds to this residue has largely remained unknown. In this study, we analyzed the role of adaptor complexes in the polarized sorting of APP. We show that the medium subunit μ1B of the epithelia‐specific adaptor protein (AP)‐1B binds onto the cytoplasmic tail of APP in a Tyr653‐dependent way. Moreover, ectopic expression of μ1B in cells lacking AP‐1B resulted in correction of apical missorting of wild‐type but not Tyr653Ala APP. Basolateral secretion of sAPP was found to be independent of Tyr653. We propose a model for polarized targeting of APP according to which sorting of APP to basolateral domain is dependent on binding of AP‐1B on Tyr653 in basolateral endosomes. This model is in accordance with the current understanding of sorting mechanisms mediating polarized targeting of membrane proteins.

A mutation in the signal sequence of LRP5 in a family with an osteoporosis-pseudoglioma syndrome (OPPG)-like phenotype indicates a novel disease mechanism for trinucleotide repeats.

We extend the spectrum of phenotypes caused by mutations in the Wnt/Norrin coreceptor low‐density lipoprotein receptor‐related protein 5 (LRP5) by identifying two novel types of mutation in related individuals whose presenting features were profound muscle hypotonia, mild mental retardation, blindness, and growth retardation. One mutation removes 6 out of 9 consecutive leucine residues in the LRP5 signal peptide (c.43_60del or p.Leu15_Leu20del), which impairs polypeptide entry into the endoplasmic reticulum (ER), trafficking to the cell membrane, and signal transduction. The second mutation resulted from nonhomologous recombination between Alu repeat sequences, which deleted exons 14–16 and would produce a nonfunctional, truncated, and frameshifted polypeptide, if expressed [chr11:g.(13871447_1387511)_(13879636_13879700)del (NW_925106.1) or p.Pro1010GlnfsX38]. We confirmed that the length of the LRP5 signal peptide poly‐leucine repeat is polymorphic in the general population, and, importantly, we were able to demonstrate in independent in vitro assays that different allele sizes affect receptor processing and signal transduction. Consequently, this polymorphism may have physiologic effects in vivo. This latter finding is relevant since through a genomewide search we identified nearly 400 human proteins that contain poly‐leucine repeats within their signal peptide. We chose 18 of these proteins and genotyped the underlying trinucleotide repeat in healthy Caucasian individuals. More than one length allele was observed in one‐half of the proteins. We therefore propose that natural variation in poly‐leucine‐stretches within signal peptides constitutes a currently unrecognized source of variability in protein translation and expression. Hum Mutat 0, 1–8, 2009.

Cutting Edge: The UNC93B1 Tyrosine-Based Motif Regulates Trafficking and TLR Responses via Separate Mechanisms

Sensing of nucleic acids by TLRs is crucial in the host defense against viruses and bacteria. Unc-93 homolog B1 (UNC93B1) regulates the trafficking of nucleic acid–sensing TLRs from the endoplasmic reticulum to endolysosomes, where the TLRs encounter their respective ligands and become activated. In this article, we show that a carboxyl-terminal tyrosine-based sorting motif (YxxΦ) in UNC93B1 differentially regulates human nucleic acid–sensing TLRs in a receptor- and ligand-specific manner. Destruction of YxxΦ abolished TLR7, TLR8, and TLR9 activity toward nucleic acids in human B cells and monocytes, whereas TLR8 responses toward small molecules remained intact. YxxΦ in UNC93B1 influenced the subcellular localization of human UNC93B1 via both adapter protein complex (AP)1- and AP2-dependent trafficking pathways. However, loss of AP function was not causal for altered TLR responses, suggesting AP-independent functions of YxxΦ in UNC93B1.