The vesicle- and target-SNARE proteins that mediate Glut4 vesicle fusion are localized in detergent-insoluble lipid rafts present on distinct intracellular membranes

Insulin stimulates the fusion of intracellular vesicles containing the glucose transporter Glut4 with the plasma membrane in adipocytes and muscle cells. Glut4 vesicle fusion is thought to be catalyzed by the interaction of the vesicle soluble N-ethyl-maleimide-sensitive fusion protein attachment protein receptor VAMP2 with the target soluble N-ethyl-maleimide-sensitive fusion protein attachment protein receptors SNAP-23 and syntaxin 4. Here, we use combined membrane fractionation, detergent solubility, and sucrose gradient flotation to demonstrate that the large majority (>70%) of SNAP-23 and a significant proportion of syntaxin 4 ( approximately 35%) are associated with plasma membrane lipid rafts in 3T3-L1 adipocytes. Furthermore, VAMP2 is shown to be concentrated in lipid rafts isolated from intracellular membranes. Insulin stimulation had no effect on the plasma membrane raft association of SNAP-23 or syntaxin 4 but promoted VAMP2 insertion into plasma membrane rafts. Immunofluorescence analysis revealed that SNAP-23 was clustered at the plasma membrane and almost completely segregated from the transferrin receptor. SNAP-23 distribution seemed to be distinct from caveolin-1, and clusters of SNAP-23 were dispersed after cholesterol extraction with methyl-beta-cyclodextrin, suggesting that the majority of SNAP-23 is associated with non-caveolar, cholesterol-rich lipid rafts. The results described implicate lipid rafts as important platforms for Glut4 vesicle fusion and suggest the hypothesis that such rafts may represent a spatial integration point of insulin signaling and membrane traffic.     

Evaluation of C-H cdots,three dots,centered O hydrogen bonds in native and misfolded proteins

Non-traditional C-H cdots, three dots, centered Y hydrogen bonds, in which a carbon atom acts as the hydrogen donor and an electronegative atom Y (Y=N, O or S) acts as the acceptor, have been reported in proteins, but their importance in protein structures is not well established. Here, we present the results of three computational tests that examine the significance of C-H cdots, three dots, centered Y bonds involving the C(alpha) in proteins. First, we compared the number of C(alpha)-H cdots, three dots, centered Y bonds in native structures with two sets of compact, energy-minimized decoy structures. The decoy structures contain about as many C(alpha)-H cdots, three dots, centered Y bonds as the native structures, indicating that the constraints of chain connectivity and compactness can lead to incidental formation of C(alpha)-H cdots, three dots, centered Y bonds. Secondly, we examined whether short C(alpha)-H cdots, three dots, centered Y bonds have a tendency to be linear, as is expected for a cohesive hydrogen-bonding interaction. The native structures do show this trend, but so does one of the decoy sets, suggesting that this criterion is also not sufficient to indicate a stabilizing interaction. Finally, we examined the preference for C(alpha)-H cdots, three dots, centered Y bond donors to be near to strong hydrogen bond acceptors. In the native proteins, the alpha protons attract strong acceptors like oxygen atoms more than weak acceptors. In contrast, hydrogen bond donors in the decoy structures do not distinguish between strong and weak acceptors. Thus, any individual C(alpha)-H cdots, three dots, centered Y bond may be fortuitous and occur due to the polypeptide connectivity and compactness. Taken collectively, however, C(alpha)-H cdots, three dots, centered Y bonds provide a weakly cohesive force that stabilizes proteins.     

Age estimation from the auricular surface of the ilium: a revised method

A revised method for estimating adult age at death using the auricular surface of the ilium has been developed. It is based on the existing auricular surface aging method of Lovejoy et al. ([1985] Am. J. Phys. Anthropol. 68:15-28), but the revised technique is easier to apply, and has low levels of inter- and intraobserver error. The new method records age-related stages for different features of the auricular surface, which are then combined to provide a composite score from which an estimate of age at death is obtained. Blind tests of the method were carried out on a known-age skeletal collection from Christ Church, Spitalfields, London. These tests showed that the dispersion of age at death for a given morphological stage was large, particularly after the first decade of adult life. Statistical analysis showed that the age-related changes in auricular surface are not significantly different for males and females. The scores from the revised method have a slightly higher correlation with age than do the Suchey-Brooks pubic symphysis stages. Considering the higher survival rates of the auricular surface compared with the pubic symphysis, this method promises to be useful for biological anthropology and forensic science.     

Watching proteins in the wild: fluorescence methods to study protein dynamics in living cells

The advent of GFP imaging has led to a revolution in the study of live cell protein dynamics. Ease of access to fluorescently tagged proteins has led to their widespread application and demonstrated the power of studying protein dynamics in living cells. This has spurred development of next generation approaches enabling not only the visualization of protein movements, but correlation of a protein's dynamics with its changing structural state or ligand binding. Such methods make use of fluorescence resonance energy transfer and dyes that report changes in their environment, and take advantage of new chemistries for site-specific protein labeling.     

Cysteine-string protein: the chaperone at the synapse

Cysteine-string protein (Csp) is a major synaptic vesicle and secretory granule protein first discovered in Drosophila and Torpedo. Csps were subsequently identified from Xenopus, Caenorhabditis elegans, and mammalian species. It is clear from the study of a null mutant in Drosophila that Csp is required for viability of the organism and that it has a key role in neurotransmitter release. In addition, other studies have directly implicated Csp in regulated exocytosis in mammalian neuroendocrine and endocrine cell types, and its distribution suggests a general role in regulated exocytosis. An early hypothesis was that Csp functioned in the control of voltage-gated Ca2+ channels. Csp, however, must have an additional function as a direct regulator of the exocytotic machinery as changes in Csp expression modify the extent of exocytosis triggered directly by Ca2+ in permeabilised cells. Csps possess a cysteine-string domain that is highly palmitoylated and confers membrane targeting. In addition, Csps have a conserved "J" domain that mediates binding to an activation of the Hsp70/ Hsc70 chaperone ATPases. This and other evidence implicate Csps as molecular chaperones in the synapse that are likely to control the correct conformational folding of one or more components of the vesicular exocytotic machinery. Targets for Csp include the vesicle protein VAMP/synaptobrevin and the plasma membrane protein syntaxin 1, the significance of which is discussed in possible models to account for current knowledge of Csp function.     

A model of the closed form of the nicotinic acetylcholine receptor m2 channel pore

The nicotinic acetylcholine receptor is a neurotransmitter-gated ion channel in the postsynaptic membrane. It is composed of five homologous subunits, each of which contributes one transmembrane helix--the M2 helix--to create the channel pore. The M2 helix from the delta subunit is capable of forming a channel by itself. Although a model of the receptor was recently proposed based on a low-resolution, cryo-electron microscopy density map, we found that the model does not explain much of the other available experimental data. Here we propose a new model of the M2 channel derived solely from helix packing and symmetry constraints. This model agrees well with experimental results from solid-state NMR, chemical reactivity, and mutagenesis experiments. The model depicts the channel pore, the channel gate, and the residues responsible for cation specificity.