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By admin, on January 1st, 2012
Cell Mol Biol Lett. 2012 Mar; 17(1): 136-52
Rajasekharan A, Gummadi SN
Biogenic membranes or self-synthesizing membranes are the site of synthesis of new lipids such as the endoplasmic reticulum (ER) in eukaryotes. Newly synthesized phospholipids (PLs) at the cytosolic leaflet of ER need to be translocated to the lumen side for membrane biogenesis and this is facilitated by a special class of lipid translocators called biogenic membrane flippase. Even though ER is the major site of cholesterol synthesis, it contains very low amounts of cholesterol, since newly synthesized cholesterol in ER is rapidly transported to other organelles and is highly enriched in plasma membrane. Thus, only low levels of cholesterol are present at the biosynthetic compartment (ER), which results in loose packing of ER lipids. We hypothesize that the prevalence of cholesterol in biogenic membranes might affect the rapid flip-flop. To validate our hypothesis, detergent solubilized ER membranes from both bovine liver and spinach leaves were reconstituted into proteoliposomes with varying mol% of cholesterol. Our results show that (i) with increase in the cholesterol/PL ratio, the half-life time of PL translocation increased, suggesting that cholesterol affects the kinetics of flipping, (ii) flipping activity was completely inhibited in proteoliposomes reconstituted with 1 mol% cholesterol, and (iii) FRAP and DSC experiments revealed that 1 mol% cholesterol did not alter the bilayer properties significantly and that flippase activity inhibition is probably mediated by interaction of cholesterol with the protein.
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Inhibition of biogenic membrane flippase activity in reconstituted ER proteoliposomes in the presence of low cholesterol levels.
By admin, on December 24th, 2011
J Proteome Res. 2011 Dec 23;
Takahashi D, Kawamura Y, Yamashita T, Uemura M
The plasma membrane (PM) is involved in important cellular processes that determine the growth, development, differentiation and environmental signal responses of plant cells. Some of these dynamic reactions occur in specific domains in the PM. In this study, we performed comparable nano-LC-MS/MS-based large-scale proteomic analysis of detergent-resistant membrane (DRM) fractions prepared from the PM of oat and rye. A number of proteins showed differential accumulation between the PM and DRM, and some proteins were only found in the DRM. Numerous proteins were identified as DRM proteins in oat (219 proteins) and rye (213 proteins), of which about half were identified only in the DRM. The DRM proteins were largely common to those found in dicotyledonous plants (Arabidopsis and tobacco), suggesting common functions associated with the DRM in plants. Combination of semiquantitative proteomic analysis and prediction of post-translational protein modification sites revealed differences in several proteins associated with the DRM in oat and rye. It is concluded that protein distribution in the DRM is unique from that in the PM, partly because of the physicochemical properties of the proteins and the unique distribution of these proteins may define the functions of the specific domains in the PM in various physiological processes in plant cells.
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Detergent-resistant Plasma Membrane Proteome in Oat and Rye: Similarities and Dissimilarities between Two Monocotyledonous Plants.
By admin, on December 24th, 2011
J Proteome Res. 2011 Dec 23;
Takahashi D, Kawamura Y, Yamashita T, Uemura M
The plasma membrane (PM) is involved in important cellular processes that determine the growth, development, differentiation and environmental signal responses of plant cells. Some of these dynamic reactions occur in specific domains in the PM. In this study, we performed comparable nano-LC-MS/MS-based large-scale proteomic analysis of detergent-resistant membrane (DRM) fractions prepared from the PM of oat and rye. A number of proteins showed differential accumulation between the PM and DRM, and some proteins were only found in the DRM. Numerous proteins were identified as DRM proteins in oat (219 proteins) and rye (213 proteins), of which about half were identified only in the DRM. The DRM proteins were largely common to those found in dicotyledonous plants (Arabidopsis and tobacco), suggesting common functions associated with the DRM in plants. Combination of semiquantitative proteomic analysis and prediction of post-translational protein modification sites revealed differences in several proteins associated with the DRM in oat and rye. It is concluded that protein distribution in the DRM is unique from that in the PM, partly because of the physicochemical properties of the proteins and the unique distribution of these proteins may define the functions of the specific domains in the PM in various physiological processes in plant cells.
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Detergent-resistant Plasma Membrane Proteome in Oat and Rye: Similarities and Dissimilarities between Two Monocotyledonous Plants.
By admin, on November 30th, 2011
By admin, on November 30th, 2011
By admin, on November 24th, 2011
PLoS One. 2011; 6(11): e27884
Xu L, Pallikkuth S, Hou Z, Mignery GA, Robia SL, Han R
Dysferlin was previously identified as a key player in muscle membrane repair and its deficiency leads to the development of muscular dystrophy and cardiomyopathy. However, little is known about the oligomerization of this protein in the plasma membrane. Here we report for the first time that dysferlin forms a dimer in vitro and in living adult skeletal muscle fibers isolated from mice. Endogenous dysferlin from rabbit skeletal muscle exists primarily as a ∼460 kDa species in detergent-solubilized muscle homogenate, as shown by sucrose gradient fractionation, gel filtration and cross-linking assays. Fluorescent protein (YFP) labeled human dysferlin forms a dimer in vitro, as demonstrated by fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analyses. Dysferlin also dimerizes in living cells, as probed by fluorescence resonance energy transfer (FRET). Domain mapping FRET experiments showed that dysferlin dimerization is mediated by its transmembrane domain and by multiple C2 domains. However, C2A did not significantly contribute to dimerization; notably, this is the only C2 domain in dysferlin known to engage in a Ca-dependent interaction with cell membranes. Taken together, the data suggest that Ca-insensitive C2 domains mediate high affinity self-association of dysferlin in a parallel homodimer, leaving the Ca-sensitive C2A domain free to interact with membranes.
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Dysferlin forms a dimer mediated by the c2 domains and the transmembrane domain in vitro and in living cells.
By admin, on November 21st, 2011
Exploitation of detergent thermodynamics in the direct solubilization of myelin membrane proteins for two-dimensional gel electrophoresis for proteomic analysis. by Sreepriya Nair on November 21, 2011 …
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Exploitation of detergent thermodynamics in the direct solubilization …
By admin, on November 21st, 2011
Exploitation of detergent thermodynamics in the direct solubilization of myelin membrane proteins for two-dimensional gel electrophoresis for proteomic analysis. by Sreepriya Nair on November 21, 2011 …
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Exploitation of detergent thermodynamics in the direct solubilization …
By admin, on November 15th, 2011
J Bacteriol. 2011 Nov 11;
Zou SB, Hersch SJ, Roy H, Wiggers JB, Leung AS, Buranyi S, Xie JL, Dare K, Ibba M, Navarre WW
EF-P is post-translationally modified at a conserved lysyl residue by the coordinated action of two enzymes, PoxA and YjeK. We have previously established the importance of this modification in Salmonella stress resistance. Here we report that, like poxA and yjeK mutants, Salmonella strains lacking EF-P display increased susceptibility to hypoosmotic conditions, antibiotics, detergents and enhanced resistance to the compound S-nitrosoglutathione (GSNO). The susceptibility phenotypes are largely explained by the enhanced membrane permeability of the efp mutant, which exhibits increased uptake of the hydrophobic dye 1-N-phenylnapthylamine (NPN). Analysis of the membrane proteome in wild-type and efp mutant Salmonella reveals few changes, including a prominent overexpression of a single porin, KdgM, in the efp mutant outer membrane. Removal of KdgM in the efp mutant background ameliorates the detergent, antibiotic, and osmosensitivity phenotypes and restores wild-type permeability to NPN. Our data support a role for EF-P in the translational regulation of a limited number of proteins that, when perturbed, renders the cell susceptible to stress by the adventitious overexpression of an outer membrane porin.
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Loss of Elongation factor P Disrupts Bacterial Outer Membrane Integrity.
By admin, on November 10th, 2011
Proc Natl Acad Sci U S A. 2011 Sep 13; 108(37): 15147-51
Smirnova I, Kasho V, Sugihara J, Kaback HR
The lactose permease (LacY) catalyzes galactoside/H(+) symport via an alternating access mechanism in which sugar- and H(+)-binding sites in the middle of the molecule are alternatively exposed to either side of the membrane by opening and closing of inward- and outward-facing cavities. The crystal structures of wild-type LacY, as well as accessibility data for the protein in the membrane, provide strong support for a conformation with a tightly closed periplasmic side and an open cytoplasmic side (an inward-facing conformation). In this study, rates of substrate binding were measured by stopped-flow with purified LacY either in detergent or in reconstituted proteoliposomes. Binding rates are compared with rates of sugar-induced opening of the periplasmic pathway obtained by using a recently developed method based on unquenching of Trp fluorescence. A linear dependence of galactoside-binding rates on sugar concentration is observed in detergent, whereas reconstituted LacY binds substrate at a slower rate that is independent of sugar concentration. Rates of opening of the periplasmic cavity with LacY in detergent are independent of substrate concentration and are essentially the same for different galactosidic sugars. The findings demonstrate clearly that reconstituted LacY is oriented physiologically with a closed periplasmic side that limits access of sugar to the binding site. Moreover, opening of the periplasmic cavity is the limiting factor for sugar binding with reconstituted LacY and may be the limiting step in the overall transport reaction.
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Opening the periplasmic cavity in lactose permease is the limiting step for sugar binding.
By admin, on November 10th, 2011
Proc Natl Acad Sci U S A. 2011 Sep 13; 108(37): 15147-51
Smirnova I, Kasho V, Sugihara J, Kaback HR
The lactose permease (LacY) catalyzes galactoside/H(+) symport via an alternating access mechanism in which sugar- and H(+)-binding sites in the middle of the molecule are alternatively exposed to either side of the membrane by opening and closing of inward- and outward-facing cavities. The crystal structures of wild-type LacY, as well as accessibility data for the protein in the membrane, provide strong support for a conformation with a tightly closed periplasmic side and an open cytoplasmic side (an inward-facing conformation). In this study, rates of substrate binding were measured by stopped-flow with purified LacY either in detergent or in reconstituted proteoliposomes. Binding rates are compared with rates of sugar-induced opening of the periplasmic pathway obtained by using a recently developed method based on unquenching of Trp fluorescence. A linear dependence of galactoside-binding rates on sugar concentration is observed in detergent, whereas reconstituted LacY binds substrate at a slower rate that is independent of sugar concentration. Rates of opening of the periplasmic cavity with LacY in detergent are independent of substrate concentration and are essentially the same for different galactosidic sugars. The findings demonstrate clearly that reconstituted LacY is oriented physiologically with a closed periplasmic side that limits access of sugar to the binding site. Moreover, opening of the periplasmic cavity is the limiting factor for sugar binding with reconstituted LacY and may be the limiting step in the overall transport reaction.
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Opening the periplasmic cavity in lactose permease is the limiting step for sugar binding.
By admin, on November 9th, 2011
J Membr Biol . 2011 Nov 6; Godbole A, Mitra R, Dubey AK, Reddy PS, Mathew MK The voltage-dependent anion-selective channel (VDAC) is the most abundant protein in the mitochondrial outer membrane and forms the major conduit for metabolite transport across this membrane. VDACs from different sources show varied primary sequence but conserved functional properties
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Bacterial Expression, Purification and Characterization of a Rice Voltage-Dependent, Anion-Selective Channel Isoform, OsVDAC4.
By admin, on November 5th, 2011
J Reprod Dev. 2011 Nov 4;
Mori E, Fukuda H, Imajoh-Ohmi S, Mori T, Takasaki S
Although the importance of carbohydrate recognition by sperm during egg zona pellucida binding has been widely reported, the sperm molecular species that recognize the carbohydrates are poorly characterized. Our previous cytochemical study indicated that two kinds of carbohydrate-binding proteins are expressed on porcine sperm heads-one recognizes N-acetyllactosamine (Galβ1-4GlcNAc-), and the other recognizes the Lewis X structure (Galβ1-4(Fucα1-3)GlcNAc-). For this report, we used proteomic techniques to characterize the sperm proteins that bind N-acetyllactosamine. Porcine sperm plasma membrane was solubilized with a detergent solution and subjected to sequential chromatography with dextran sulfate agarose, affinity, and hydroxyapatite, and the binding activities in the eluates were monitored by a solid-phase binding assay. The tryptic peptides of two proteins most likely associated with the binding activities were subjected to tandem mass spectrometry sequencing. A subsequent database search identified one of the two proteins as predicted disintegrin and metalloprotease domain-containing protein 20-like (XP_003128672). The other protein was identified as disintegrin and metalloprotease domain-containing protein 5 (AB613817) by database searches for homologous amino acid sequences, cDNA cloning, nucleotide sequencing and nucleotide database searches. Furthermore, two-dimensional blue native/SDS-PAGE demonstrated that they formed a variety of non-covalent complexes. Therefore, these ADAM complexes probably are responsible for the N-acetyllactosamine-binding activity. An affinity-purified fraction containing these ADAM complexes showed zona pellucida-binding activity, though the activity was relatively weak, and the presence of another zona pellucida-binding protein that probably works in concert with these ADAM complexes was suggested. Immunofluorescence testing suggested that ADAM20-like was localized on the anterior part of the sperm plasma membrane.
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Purification of N-acetyllactosamine-binding Activity from the Porcine Sperm Membrane: Possible Involvement of an ADAM Complex in the Carbohydrate-binding Activity of Sperm.
By admin, on November 1st, 2011
By admin, on November 1st, 2011
By admin, on November 1st, 2011
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