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.

0bjective:To estimate intratester and intertester reliability and learning effects during the Star Excursion Balance Tests (SEBTs).Setting:A university athletic training research laboratory.Subjects:Sixteen healthy volunteers with no history of balance disorders or significant lower extremity joint pathology.Measurements:Length of excursion was measured manually for each trial.Results:ICCs for intratester reliability were .78–.96 on day 1 and 32–.96 on day 2. ICCs for intertester reliability were .35–.84 on day 1 and .81–.93 on day 2. Significant learning effects were identified for 4 of the 8 tests.Conclusions:Estimates of intratester and intertester reliability were high, but adequate practice trials should be performed before taking baseline measures.

SummarySNAREs 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, PtdIns4,5P2-binding, endocytic clathrin adaptor CALM/PICALM. X-ray crystallography shows that the N-terminal halves of their SNARE motifs bind the CALMANTH 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. CALM's 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 Alzheimer's disease.

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.