The mechanisms underlying sterol transport in mammalian cells are poorly understood. In particular, how cholesterol internalized from HDL is made available to the cell for storage or modification is unknown. Here, we describe three ER-resident proteins (Aster-A, -B, -C) that bind cholesterol and facilitate its removal from the plasma membrane. The crystal structure of the central domain of Aster-A broadly resembles the sterol-binding fold of mammalian StARD proteins, but sequence differences in the Aster pocket result in a distinct mode of ligand binding. The Aster N-terminal GRAM domain binds phosphatidylserine and mediates Aster recruitment to plasma membrane-ER contact sites in response to cholesterol accumulation in the plasma membrane. Mice lacking Aster-B are deficient in adrenal cholesterol ester storage and steroidogenesis because of an inability to transport cholesterol from SR-BI to the ER. These findings identify a nonvesicular pathway for plasma membrane to ER sterol trafficking in mammals.
Half the world's population is chronically infected with Helicobacter pylori1, causing gastritis, ulcers and increased incidence of gastric adenocarcinoma2. Its proton-gated inner-membrane urea channel, HpUreI, is essential for survival in the acidic environment of the stomach3. The channel is closed at neutral pH and opens at acidic pH to allow rapid urea access to cytoplasmic urease4. Urease produces NH3 and CO2 that neutralize entering protons and thus buffer the periplasm to pH ∼6.1 even in gastric juice at pH <2.0. Here we report the structure of HpUreI, revealing six protomers assembled in a hexameric ring surrounding a central bilayer plug of ordered lipids. Each protomer encloses a channel formed by a twisted bundle of six transmembrane helices. The bundle defines a novel fold comprising a two-helix hairpin motif repeated three times around the central axis of the channel, without the inverted repeat of mammalian urea transporters. Both the channel and the protomer interface contain residues conserved in the AmiS/UreI superfamily, suggesting preservation of channel architecture and oligomeric state in this superfamily. Predominantly aromatic or aliphatic side chains line the entire channel and define two consecutive constriction sites in the middle of the channel. Mutation of Trp153 in the cytoplasmic constriction site to Ala or Phe reduces the selectivity for urea compared to thiourea, suggesting that solute interaction with Trp153 contributes specificity. The novel hexameric channel structure described here provides a new paradigm for permeation of urea and other small amide solutes in prokaryotes and archaea.
Na؉ ,K ؉ -ATPase (pig ␣1,1) has been expressed in the methylotrophic yeast Pichia pastoris. A protease-deficient strain was used, recombinant clones were screened for multicopy genomic integrants, and protein expression, and time and temperature of methanol induction were optimized. A 3-liter culture provides 300 -500 mg of membrane protein with ouabain binding capacity of 30 -50 pmol mg ؊1 . The Na ϩ ,K ϩ -ATPase utilizes the free energy of hydrolysis of ATP to actively transport three intracellular Na ϩ ions and two extracellular K ϩ ions in opposite directions across animal cell membranes. The Na ϩ ,K ϩ -ATPase is a member of the P-type family of cation pumps. The kinetic mechanism of Na ϩ ,K ϩ -ATPase, as of other P-type pumps, involves a phosphoenzyme intermediate and is now largely understood (1, 2). As pointed out by Jencks (3), strict cation and substrate specificities of the phosphorylation and dephosphorylation reactions, and tight coupling of the E 1 7 E 2 conformational changes to cation movements are the essential features of all P-type ion pump mechanisms. These central questions of the energy transduction mechanism of P-type pumps can now be posed in structural terms (4) because of availability of molecular structures of the sarcoplasmic reticulum Ca 2ϩ -ATPase for both E 1 2Ca 2ϩ and E 2 conformations (5, 6).The Ca 2ϩ -ATPase molecule consists of head, stalk, and membrane sectors (5). There are 10 transmembrane segments in the membrane domain with two Ca 2ϩ ions ligated approximately in the center of the bilayer and between transmembrane segments M4, M5, M6, and M8 in the E 1 2Ca 2ϩ conformation. The stalk sector consists of the cytoplasmic extensions of the transmembrane helices, particularly S5 and S4. The cytoplasmic sector consists of three domains, nucleotide binding (N), 1 phosphorylating (P), and anchor or actuator domain (A). Comparison of the crystal structure an E 1 2Ca2ϩ and E 2 conformations shows that in the E 1 conformations the N, P, and A domains are separate, whereas in E 2 conformations the domains are gathered together, moving essentially as rigid bodies (5, 6). Movement of the A domain toward P and N domains in the E 1 3 E 2 transition is associated with a bending of S5 that entails complex movements of several transmembrane segments. This changes the ligation of the occluded Ca 2ϩ ions within the transmembrane segments allowing them to dissociate within the sarcoplasmic reticulum. Whereas this general paradigm clearly applies to the other P-type pumps, not all features are explained by the crystal structures. As one example, phosphorylation by ATP requires close proximity of the nucleotide binding N and phosphorylation P domains, but this is not observed in the E 1 2Ca 2ϩ (Protein Data Bank code 1EUL) structure. In addition, of course, there are the specific features of other ion pumps particularly the cation selectivities,
We isolated from the venom of the scorpion Leiurus quinquestriatus hebraeus an extremely active anti-insect selective depressant toxin, Lqh-dprIT(3). Cloning of Lqh-dprIT(3) revealed a gene family encoding eight putative polypeptide variants (a-h) differing at three positions (37A/G, 50D/E, and 58N/D). All eight toxin variants were expressed in a functional form, and their toxicity to blowfly larvae, binding affinity for cockroach neuronal membranes, and CD spectra were compared. This analysis links Asn-58, which appears in variants a-d, to a toxin conformation associated with high binding affinity for insect sodium channels. Variants e-h, bearing Asp-58, exhibit a different conformation and are less potent. The importance of Asn-58, which is conserved in other depressant toxins, was further validated by construction and analysis of an N58D mutant of the well-characterized depressant toxin, LqhIT(2). Current and voltage clamp assays using the cockroach giant axon have shown that despite the vast difference in potency, the two types of Lqh-dprIT(3) variants (represented by Lqh-dprIT(3)-a and Lqh-dprIT(3)-e) are capable of blocking the action potentials (manifested as flaccid paralysis in blowfly larvae) and shift the voltage dependence of activation to more negative values, which typify the action of beta-toxins. Moreover, the stronger and faster shift in voltage dependence of activation and lack of tail currents observed in the presence of Lqh-dprIT(3)-a suggest an extremely efficient trapping of the voltage sensor compared to that of Lqh-dprIT(3)-e. The current clamp assays revealed that repetitive firing of the axon, which is reflected in contraction paralysis of blowfly larvae, can be obtained with either the less potent Lqh-dprIT(3)-e or the highly potent Lqh-dprIT(3)-a at more negative membrane potentials. Thus, the contraction symptoms in flies are likely to be dominated by the resting potential of neuronal membranes. This study clarifies the electrophysiological basis of the complex symptoms induced by scorpion depressant toxins in insects, and highlights for the first time molecular features involved in their activity.
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