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Signal sequence insufficiency contributes to neurodegeneration caused by transmembrane prion protein
Neena S. Rane Oishee Chakrabarti Lionel Feigenbaum Ramanujan S. Hegde 《The Journal of cell biology》2010,188(4):515-526
Protein translocation into the endoplasmic reticulum is mediated by signal sequences that vary widely in primary structure. In vitro studies suggest that such signal sequence variations may correspond to subtly different functional properties. Whether comparable functional differences exist in vivo and are of sufficient magnitude to impact organism physiology is unknown. Here, we investigate this issue by analyzing in transgenic mice the impact of signal sequence efficiency for mammalian prion protein (PrP). We find that replacement of the average efficiency signal sequence of PrP with more efficient signals rescues mice from neurodegeneration caused by otherwise pathogenic PrP mutants in a downstream hydrophobic domain (HD). This effect is explained by the demonstration that efficient signal sequence function precludes generation of a cytosolically exposed, disease-causing transmembrane form of PrP mediated by the HD mutants. Thus, signal sequences are functionally nonequivalent in vivo, with intrinsic inefficiency of the native PrP signal being required for pathogenesis of a subset of disease-causing PrP mutations. 相似文献
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Jelena Baranovic Chandra S. Ramanujan Nahoko Kasai Charles R. Midgett Dean R. Madden Keiichi Torimitsu John F. Ryan 《The Journal of biological chemistry》2013,288(12):8647-8657
AMPA receptors (AMPARs) are glutamate-gated ion channels ubiquitous in the vertebrate central nervous system, where they mediate fast excitatory neurotransmission and act as molecular determinants of memory formation and learning. Together with detailed analyses of individual AMPAR domains, structural studies of full-length AMPARs by electron microscopy and x-ray crystallography have provided important insights into channel assembly and function. However, the correlation between the structure and functional states of the channel remains ambiguous particularly because these functional states can be assessed only with the receptor bound within an intact lipid bilayer. To provide a basis for investigating AMPAR structure in a membrane environment, we developed an optimized reconstitution protocol using a receptor whose structure has previously been characterized by electron microscopy. Single-channel recordings of reconstituted homomeric GluA2flop receptors recapitulate key electrophysiological parameters of the channels expressed in native cellular membranes. Atomic force microscopy studies of the reconstituted samples provide high-resolution images of membrane-embedded full-length AMPARs at densities comparable to those in postsynaptic membranes. The data demonstrate the effect of protein density on conformational flexibility and dimensions of the receptors and provide the first structural characterization of functional membrane-embedded AMPARs, thus laying the foundation for correlated structure-function analyses of the predominant mediators of excitatory synaptic signals in the brain. 相似文献
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Lingappa VR Rutkowski DT Hegde RS Andersen OS 《BioEssays : news and reviews in molecular, cellular and developmental biology》2002,24(8):741-748
We suggest a new view of secretory and membrane protein folding that emphasizes the role of pathways of biogenesis in generating functional and conformational heterogeneity. In this view, heterogeneity results from action of accessory factors either directly binding specific sequences of the nascent chain, or indirectly, changing the environment in which a particular domain is synthesized. Entrained by signaling pathways, these variables create a combinatorial set of necessary-but-not-sufficient conditions that enhance synthesis and folding of particular alternate, functional, conformational forms. We therefore propose that protein conformation is productively regulated by the cell during translocation across the endoplasmic reticulum (ER), a concept that may account for currently poorly understood aspects of physiological function, natural selection, and disease pathogenesis. 相似文献
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AFM observation of single,functioning ionotropic glutamate receptors reconstituted in lipid bilayers
Nahoko Kasai Chandra S. Ramanujan Ichiro Fujimoto Akiyoshi Shimada John F. Ryan Keiichi Torimitsu 《Biochimica et Biophysica Acta (BBA)/General Subjects》2010
Background
Ionotropic glutamate receptors (iGluRs) are responsible for extracellular signaling in the central nervous system. However, the relationship between the overall structure of the protein and its function has yet to be resolved. Atomic force microscopy (AFM) is an important technique that allows nano-scale imaging in liquid. In the present work we have succeeded in imaging by AFM of the external features of the most common iGluR, AMPA-R (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor), in a physiological environment.Methods
Homomeric GluR3 receptors were over-expressed in insect cells, purified and reconstituted into lipid membranes. AFM images were obtained in a buffer from membranes immobilized on a mica substrate.Results
Using Au nanoparticle-conjugated antibodies, we show that proteins reconstitute predominantly with the N-terminal domain uppermost on the membrane. A tetrameric receptor structure is clearly observed, but it displays considerable heterogeneity, and the dimensions differ considerably from cryo-electron microscopy measurements.Conclusions
Our results indicate that the extracellular domains of AMPA-R are highly flexible in a physiological environment.General significance
AFM allows us to observe the protein surface structure, suggesting the possibility of visualizing real time conformational changes of a functioning protein. This knowledge may be useful for neuroscience as well as in pharmaceutical applications. 相似文献7.
Identification of a targeting factor for posttranslational membrane protein insertion into the ER 总被引:1,自引:0,他引:1
Hundreds of proteins are anchored in intracellular membranes by a single transmembrane domain (TMD) close to the C terminus. Although these tail-anchored (TA) proteins serve numerous essential roles in cells, components of their targeting and insertion pathways have long remained elusive. Here we reveal a cytosolic TMD recognition complex (TRC) that targets TA proteins for insertion into the ER membrane. The highly conserved, 40 kDa ATPase subunit of TRC (which we termed TRC40) was identified as Asna-1. TRC40/Asna-1 interacts posttranslationally with TA proteins in a TMD-dependent manner for delivery to a proteinaceous receptor at the ER membrane. Subsequent release from TRC40/Asna-1 and insertion into the membrane depends on ATP hydrolysis. Consequently, an ATPase-deficient mutant of TRC40/Asna-1 dominantly inhibited TA protein insertion selectively without influencing other translocation pathways. Thus, TRC40/Asna-1 represents an integral component of a posttranslational pathway of membrane protein insertion whose targeting is mediated by TRC. 相似文献
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Transmembrane topogenesis of a tail-anchored protein is modulated by membrane lipid composition 下载免费PDF全文
Brambillasca S Yabal M Soffientini P Stefanovic S Makarow M Hegde RS Borgese N 《The EMBO journal》2005,24(14):2533-2542
A large class of proteins with cytosolic functional domains is anchored to selected intracellular membranes by a single hydrophobic segment close to the C-terminus. Although such tail-anchored (TA) proteins are numerous, diverse, and functionally important, the mechanism of their transmembrane insertion and the basis of their membrane selectivity remain unclear. To address this problem, we have developed a highly specific, sensitive, and quantitative in vitro assay for the proper membrane-spanning topology of a model TA protein, cytochrome b5 (b5). Selective depletion from membranes of components involved in cotranslational protein translocation had no effect on either the efficiency or topology of b5 insertion. Indeed, the kinetics of transmembrane insertion into protein-free phospholipid vesicles was the same as for native ER microsomes. Remarkably, loading of either liposomes or microsomes with cholesterol to levels found in other membranes of the secretory pathway sharply and reversibly inhibited b5 transmembrane insertion. These results identify the minimal requirements for transmembrane topogenesis of a TA protein and suggest that selectivity among various intracellular compartments can be imparted by differences in their lipid composition. 相似文献
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Numerous proteins targeted for the secretory pathway are increasingly implicated in functional or pathological roles at alternative cellular destinations. The parameters that allow secretory or membrane proteins to reside in intracellular locales outside the secretory pathway remain largely unexplored. In this study, we have used an extremely sensitive and quantitative assay to measure the in vivo efficiency of signal sequence-mediated protein segregation into the secretory pathway. Our findings reveal that segregation efficiency varies tremendously among signals, ranging from >95 to <60%. The nonsegregated fraction is generated by a combination of mechanisms that includes inefficient signal-mediated translocation into the endoplasmic reticulum and leaky ribosomal scanning. The segregation efficiency of some, but not other signal sequences, could be influenced in cis by residues in the mature domain or in trans by yet unidentified cellular factors. These findings imply that protein compartmentalization can be modulated in a substrate-specific manner to generate biologically significant quantities of cytosolically available secretory and membrane proteins. 相似文献