Early endosomes represent the first sorting station for vesicular ubiquitylated cargo. Tollip, through its C2 domain, associates with endosomal phosphatidylinositol 3-phosphate (PtdIns(3)P) and binds ubiquitylated cargo in these compartments via its C2 and CUE domains. Tom1, through its GAT domain, is recruited to endosomes by binding to the Tollip Tom1-binding domain (TBD) through an unknown mechanism. Nuclear magnetic resonance data revealed that Tollip TBD is a natively unfolded domain that partially folds at its N terminus when bound to Tom1 GAT through high-affinity hydrophobic contacts. Furthermore, this association abrogates binding of Tollip to PtdIns(3)P by additionally targeting its C2 domain. Tom1 GAT is also able to bind ubiquitin and PtdIns(3)P at overlapping sites, albeit with modest affinity. We propose that association with Tom1 favors the release of Tollip from endosomal membranes, allowing Tollip to commit to cargo trafficking.

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The translationally-controlled tumor protein (TCTP) is a highly conserved, ubiquitously expressed, abundant protein that is broadly distributed among eukaryotes. Its biological function spans numerous cellular processes ranging from regulation of the cell cycle and microtubule stabilization to cell growth, transformation, and death processes. In this work, we propose a new function for TCTP as a “buffer protein” controlling cellular homeostasis. We demonstrate that binding of hemin to TCTP is mediated by a conserved His-containing motif (His76His77) followed by dimerization, an event that involves ligand-mediated conformational changes and that is necessary to trigger TCTP's cytokine-like activity. Mutation in both His residues to Ala prevents hemin from binding and abrogates oligomerization, suggesting that the ligand site localizes at the interface of the oligomer. Unlike heme, binding of Ca2+ ligand to TCTP does not alter its monomeric state; although, Ca2+ is able to destabilize an existing TCTP dimer created by hemin addition. In agreement with TCTP's proposed buffer function, ligand binding occurs at high concentration, allowing the “buffer” condition to be dissociated from TCTP's role as a component of signal transduction mechanisms.

observation that synaptic strength correlates with dendritic spine morphology leads to the hypothesis that the mushroom-like shape of dendritic spines functions as a receptor trap. We developed a mimetic system to investigate dendritic spine morphology and its effects on receptor confinement and diffusion. Giant unilamellar vesicles (GUV's) are made from lipids using electroswelling. To mimic the mushroom-shaped morphologies of dendritic spines, a micromanipulator is used to pull membrane tubes from the GUV lipid bilayer. Trapping capabilities for different spine morphologies are assessed by tracking quantum dots attached to membrane lipids, thus mimicking receptors. Results show a strong dependence of escape times on GUV morphology, as quantified by GUV radius and tube length. Instead of a trivial quadratic dependence of escape times on GUV radius we find a powerlaw dependence with an exponent of 2.85. This confirms the idea that receptors can be trapped by the morphology of a dendritic spine. Therefore the connection strength of a mushroom-shaped dendritic spine is much more stable than the strengths of stubby shaped dendritic spines.

In many protein ion channels found in biological membranes, a central cavity is formed between the membrane-spanning subunits of the protein, though which ions flow selectively. There is no general consensus regarding whether nonpolar cavities inside proteins are water filled or empty, or what conditions are necessary and sufficient for these two cases. The potassium channel from the soil bacteria Streptomyces lividans named KcsA, is the first of the potassiumselective channel proteins whose structure has been solved crystallographically (Doyle, D.A. et al., Science 280, 1998). KcsA has become the principal model for the pore domain in voltage-gated K þ , Na þ , and Ca 2þ channels due to highly conserved structural motifs in the pore domain characteristic for these channels. The cavity dimensions for KcsA inferred from crystallographic structures allow lessthan 20 water molecules to be accommodated. Crystallographic maps, however, do not typically reveal the true conformation of the channel in a fluid lipid environment, nor can they reveal water in cavities due to its high thermal disorder and low scattering density. In this report, we present neutron diffraction data on KcSA pore domain incorporated in lipid liquid crystalline phases. Hydrogen to Deuterium substitution is used to determine the amount of water and its distribution across the pore domain of the channel.