Protein enrichment, plasma membrane

Assemble the following reaction 20 tl nAChR-enriched plasma membranes (800 pg/ml protein 1.6 pM receptor),... 

Determination of unspecific binding reaction by competition of the RNA aptamers with cocaine 79 pi incubation buffer, 10 pi nAChR-enriched plasma membranes (800 pg/ml protein 1.6 pM receptor), 1 pi P-RNA dilution containing 10 mg/ml t-RNA anti-RNAse (40 U/pl), and 10 pi cocaine (10 mM). The percentage of binding of the ( P) RNA aptamers to the... 

Mattow J, Siejak F, Hagens K et al (2007) An improved strategy for selective and efficient enrichment of integral plasma membrane proteins of mycobacteria. Proteomics 7 1687-1701... 

Transmission of extracellular signals to the cell interior is based on receptor-induced recruitment and assembly of proteins into signaling complexes at the inner leaflet of the plasma membrane. Protein-protein and protein-lipid interactions play a crucial role in the process in which molecular proximity in specially formed membrane subdomains provides the special and temporal constraints that are required for proper signaling. The phospholipid bilayer is not merely a passive hydrophobic medium for this assembly process, but is also a site where the lipid and the protein components are enriched by a dynamic process (see Chapter 5). 

Figure 13.3 A strategy for enrichment of plasma membrane proteins using...

Microparticles are naturally enriched in plasma membrane proteins. They correspond themselves to a sub-proteome, and it is tempting to use them as a substitute to... 

In order to validate the hypothesis that microparticles are structures enriched in plasma membrane proteins, we have analysed the proteome composition of different microparticle preparations obtained from a T-Lymphocytic cell line. Such microparticles can be produced and enriched in vitro by mitogenic activation (PHA) or apoptosis induction (Act Dl, TNFo ) for instance. Microparticles protein mixtures obtained from the two stimulations were separated independently on a ID gel. The different gels were cut each 2 mm and the different slides were digested and analyzed by nanoLC-MS/MS. 

Transmission of extracellular signals to the interior of the cell is dependent on receptor-mediated assembly of proteins into signaling complexes at the inner side of the plasma membrane. Protein-protein and protein-lipid interactions are essential in these processes, whereby molecular interaction and proximity provide the spatial and temporal conditions for signaling. Tlie hydrophobic phospholipid bilayer is part of the dynamic processes of enrichment and modulation of lipid components. Utilization of fluorescent proteins interacting witli lipid components allows the analysis of molecular proximity in intact cells and sheds light on the membrane dynamics of signaling processes. 

In addition to secretory cells, many non-secretory cells are capable of regulating exocytotic fusion of transport vesicles that are derived from endosomal precursors. For instance, vesicles enriched in plasma membrane transport proteins are incorporated in a regulated manner in order to alter metabolite fluxes. Examples include the glucose transporter GLUT-4 in muscle and fat tissues, a key element in the control of... 

Eukaryotic cells utilize an efficient transport system that delivers macromolecules fast and secure to their destination. In the case of the small GTP binding proteins of the Ras family the modified C-terminus seems to be sufficient for addressing the polypeptide to its target membrane (in the case of Ras itself the plasma membrane). Lipopeptides with the C-terminal structure of N-Ras (either a pen-tamer with a C-terminal carboxymethylation and farnesylation or a heptapeptide with a palmitoyl thioester in addition) and a N-terminal 7-nitrobenz-2-oxa-l,3-diazolyl (NBD) fluorophore were microin-jected into NIH3T3 fibroblast cells and the distribution of the fluorophore was monitored by confocal laser fluorescence microscopy. Enrichment of the protein in the plasma membrane was efficient only for peptides with two hydrophobic modification sites, while the farnesylated but not palmitoylated peptide was distributed in the cytosol.1121... 

Chemical analyses of membranes isolated from various sources reveal certain common properties. Each kingdom, each species, each tissue or cell type, and the organelles of each cell type have a characteristic set of membrane lipids. Plasma membranes, for example, are enriched in cholesterol and contain no detectable cardiolipin (Fig. 11-2) in the inner mitochondrial membrane of the hepatocyte, this distribution is reversed very low cholesterol and high cardiolipin. Cardiolipin is essential to the function of certain proteins of the inner mitochondrial membrane. Cells clearly have mechanisms to control the kinds and amounts of membrane lipids they synthesize and to target specific lipids to particular organelles. In many cases, we can surmise the adaptive advantages of distinct combinations of membrane lipids in other cases, the functional significance of these combinations is as yet unknown. 

Receptor desensitisation, initiated by phosphorylation of the receptor, can be subsequently followed by receptor internalisation via multiple methods including clathrin-coated pits and/or lipid rafts/caveolae. Clathrin-coated pits are specialized regions of the cell surface that mediates the internalisation of the most of GPCRs to endosomes. Lipid rafts are planar domains in cell membranes that are enriched in specific lipid and proteins with an high content of cholesterol and glycosphingolipid (Chini and Parenti 2004). Caveolae are flask-shaped invaginations located at or near the plasma membrane and are considered a non-planar subfamily of lipid rafts (Ferguson 2001 Chini and Parenti 2004). 

More recent work suggested that one of four 4.1 proteins—4.1R—may associate with neurofilament proteins in forebrain postsynaptic densities, thus regulating the associated spectrin-rich cortex (Scott et al, 2001). Blot overlay analyses demonstrated that, in addition to spectrin and actin, postsynaptic density polypeptides included NF-L and o-internexin as interacting partners for 4.1R. Collectively, these studies emphasize that common themes are used in different cell types to both strengthen plasma membrane domains enriched in actin and IF polypeptides and to coordinate these sites with cytoplasmic architecture. 

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