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By admin, on May 16th, 2012
Dalton Trans. 2012 May 14;
Hongoh M, Haratake M, Fuchigami T, Nakayama M
In this paper, we describe a thiol-mediated and energy-dependent membrane transport of selenium by erythroid anion exchanger 1 (AE1, also known as band 3 protein). The AE1 is the most abundant integral protein of red cell membranes and plays a critical role in the carbon dioxide transport system in which carbon dioxide is carried as bicarbonate in the plasma. This protein mediates the membrane transport of selenium, an essential antioxidant micronutrient, from red cells to the plasma in a manner that is distinct from the already known anion exchange mechanism. In this pathway, selenium bound to the cysteine 93 of the hemoglobin β chain (Hb-Cysβ93) is transported by the relay mechanism to the Cys317 of the amino-terminal cytoplasmic domain of the AE1 on the basis of the intrinsic interaction between the two proteins and is subsequently exported to the plasma via the Cys843 of the membrane-spanning domain. The selenium export did not occur in plain isotonic buffer solutions and required thiols, such as albumin, in the outer medium. Such a membrane transport mechanism would also participate in the export pathways of the nitric oxide vasodilator activity and other thiol-reactive substances bound to the Hb-Cysβ93 from red cells to the plasma and/or peripherals.
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A thiol-mediated active membrane transport of selenium by erythroid anion exchanger 1 protein.
By admin, on May 16th, 2012
Dalton Trans. 2012 May 14;
Hongoh M, Haratake M, Fuchigami T, Nakayama M
In this paper, we describe a thiol-mediated and energy-dependent membrane transport of selenium by erythroid anion exchanger 1 (AE1, also known as band 3 protein). The AE1 is the most abundant integral protein of red cell membranes and plays a critical role in the carbon dioxide transport system in which carbon dioxide is carried as bicarbonate in the plasma. This protein mediates the membrane transport of selenium, an essential antioxidant micronutrient, from red cells to the plasma in a manner that is distinct from the already known anion exchange mechanism. In this pathway, selenium bound to the cysteine 93 of the hemoglobin β chain (Hb-Cysβ93) is transported by the relay mechanism to the Cys317 of the amino-terminal cytoplasmic domain of the AE1 on the basis of the intrinsic interaction between the two proteins and is subsequently exported to the plasma via the Cys843 of the membrane-spanning domain. The selenium export did not occur in plain isotonic buffer solutions and required thiols, such as albumin, in the outer medium. Such a membrane transport mechanism would also participate in the export pathways of the nitric oxide vasodilator activity and other thiol-reactive substances bound to the Hb-Cysβ93 from red cells to the plasma and/or peripherals.
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A thiol-mediated active membrane transport of selenium by erythroid anion exchanger 1 protein.
By admin, on May 2nd, 2012
Nat Cell Biol. 2012 Apr 29;
Spira F, Mueller NS, Beck G, von Olshausen P, Beig J, Wedlich-Söldner R
The plasma membrane is made up of lipids and proteins, and serves as an active interface between the cell and its environment. Many plasma-membrane proteins are laterally segregated in the plane of the membrane, but the underlying mechanisms remain controversial. Here we investigate the distribution and dynamics of a representative set of plasma-membrane-associated proteins in yeast cells. These proteins were distributed non-homogeneously in patterns ranging from distinct patches to nearly continuous networks, and these patterns were in turn strongly influenced by the lipid composition of the plasma membrane. Most proteins segregated into distinct domains. However, proteins with similar or identical transmembrane sequences (TMSs) showed a marked tendency to co-localize. Indeed we could predictably relocate proteins by swapping their TMSs. Finally, we found that the domain association of plasma-membrane proteins has an impact on their function. Our results are consistent with self-organization of biological membranes into a patchwork of coexisting domains.
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Patchwork organization of the yeast plasma membrane into numerous coexisting domains.
By admin, on May 2nd, 2012
Nat Cell Biol. 2012 Apr 29;
Spira F, Mueller NS, Beck G, von Olshausen P, Beig J, Wedlich-Söldner R
The plasma membrane is made up of lipids and proteins, and serves as an active interface between the cell and its environment. Many plasma-membrane proteins are laterally segregated in the plane of the membrane, but the underlying mechanisms remain controversial. Here we investigate the distribution and dynamics of a representative set of plasma-membrane-associated proteins in yeast cells. These proteins were distributed non-homogeneously in patterns ranging from distinct patches to nearly continuous networks, and these patterns were in turn strongly influenced by the lipid composition of the plasma membrane. Most proteins segregated into distinct domains. However, proteins with similar or identical transmembrane sequences (TMSs) showed a marked tendency to co-localize. Indeed we could predictably relocate proteins by swapping their TMSs. Finally, we found that the domain association of plasma-membrane proteins has an impact on their function. Our results are consistent with self-organization of biological membranes into a patchwork of coexisting domains.
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Patchwork organization of the yeast plasma membrane into numerous coexisting domains.
By admin, on April 27th, 2012
By admin, on April 27th, 2012
By admin, on April 22nd, 2012
PLoS One. 2012; 7(4): e33042
López-Marqués RL, Poulsen LR, Palmgren MG
Plasma membranes in eukaryotic cells display asymmetric lipid distributions with aminophospholipids concentrated in the inner leaflet and sphingolipids in the outer leaflet. This unequal distribution of lipids between leaflets is, amongst several proposed functions, hypothesized to be a prerequisite for endocytosis. P4 ATPases, belonging to the P-type ATPase superfamily of pumps, are involved in establishing lipid asymmetry across plasma membranes, but P4 ATPases have not been identified in plant plasma membranes. Here we report that the plant P4 ATPase ALA1, which previously has been connected with cold tolerance of Arabidopsis thaliana, is targeted to the plasma membrane and does so following association in the endoplasmic reticulum with an ALIS protein β-subunit.
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A Putative Plant Aminophospholipid Flippase, the Arabidopsis P4 ATPase ALA1, Localizes to the Plasma Membrane following Association with a β-Subunit.
By admin, on April 19th, 2012
PLoS One. 2012; 7(4): e34923
Day CA, Kenworthy AK
Multivalent glycolipid binding toxins such as cholera toxin have the capacity to cluster glycolipids, a process thought to be important for their functional uptake into cells. In contrast to the highly dynamic properties of lipid probes and many lipid-anchored proteins, the B-subunit of cholera toxin (CTxB) diffuses extremely slowly when bound to its glycolipid receptor GM(1) in the plasma membrane of living cells. In the current study, we used confocal FRAP to examine the origins of this slow diffusion of the CTxB/GM(1) complex at the cell surface, relative to the behavior of a representative GPI-anchored protein, transmembrane protein, and fluorescent lipid analog. We show that the diffusion of CTxB is impeded by actin- and ATP-dependent processes, but is unaffected by caveolae. At physiological temperature, the diffusion of several cell surface markers is unchanged in the presence of CTxB, suggesting that binding of CTxB to membranes does not alter the organization of the plasma membrane in a way that influences the diffusion of other molecules. Furthermore, diffusion of the B-subunit of another glycolipid-binding toxin, Shiga toxin, is significantly faster than that of CTxB, indicating that the confined diffusion of CTxB is not a simple function of its ability to cluster glycolipids. By identifying underlying mechanisms that control CTxB dynamics at the cell surface, these findings help to delineate the fundamental properties of toxin-receptor complexes in intact cell membranes.
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Mechanisms underlying the confined diffusion of cholera toxin B-subunit in intact cell membranes.
By admin, on April 17th, 2012
PLoS One. 2012; 7(4): e35373
Watts SD, Suchland KL, Amara SG, Ingram SL
Regulation of chloride gradients is a major mechanism by which excitability is regulated in neurons. Disruption of these gradients is implicated in various diseases, including cystic fibrosis, neuropathic pain and epilepsy. Relatively few studies have addressed chloride regulation in neuronal processes because probes capable of detecting changes in small compartments over a physiological range are limited.In this study, a palmitoylation sequence was added to a variant of the yellow fluorescent protein previously described as a sensitive chloride indicator (YFPQS) to target the protein to the plasma membrane (mbYFPQS) of cultured midbrain neurons. The reporter partitions to the cytoplasmic face of the cellular membranes, including the plasma membrane throughout the neurons and fluorescence is stable over 30-40 min of repeated excitation showing less than 10% decrease in mbYFPQS fluorescence compared to baseline. The mbYFPQS has similar chloride sensitivity (k(50) = 41 mM) but has a shifted pKa compared to the unpalmitoylated YFPQS variant (cytYFPQS) that remains in the cytoplasm when expressed in midbrain neurons. Changes in mbYFPQS fluorescence were induced by the GABA(A) agonist muscimol and were similar in the soma and processes of the midbrain neurons. Amphetamine also increased mbYFPQS fluorescence in a subpopulation of cultured midbrain neurons that was reversed by the selective dopamine transporter (DAT) inhibitor, GBR12909, indicating that mbYFPQS is sensitive enough to detect endogenous DAT activity in midbrain dopamine (DA) neurons.The mbYFPQS biosensor is a sensitive tool to study modulation of intracellular chloride levels in neuronal processes and is particularly advantageous for simultaneous whole-cell patch clamp and live-cell imaging experiments.
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A sensitive membrane-targeted biosensor for monitoring changes in intracellular chloride in neuronal processes.
By admin, on April 17th, 2012
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2012 Apr 1; 68(Pt 4): 424-7
Hein KL, Nissen P, Morth JP
Ca(2+)-ATPases are members of a large family of membrane proteins that maintain the selective movement of cations across biological membranes. A putative Listeria monocytogenes Ca(2+)-ATPase (Lmo0818) was crystallized in an unknown functional state. The crystal belonged to space group P2(1)2(1)2(1) and a complete data set was collected to 3.2 Å resolution. The molecular-replacement solution obtained revealed that Lmo0818 is likely to adopt an E2-like state mimicking the phosphorylated intermediate in the functional cycle of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) and a stacked bilayer `type I' packing in the crystal.
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Purification, crystallization and preliminary crystallographic studies of a PacL homologue from Listeria monocytogenes.
By admin, on April 17th, 2012
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2012 Apr 1; 68(Pt 4): 424-7
Hein KL, Nissen P, Morth JP
Ca(2+)-ATPases are members of a large family of membrane proteins that maintain the selective movement of cations across biological membranes. A putative Listeria monocytogenes Ca(2+)-ATPase (Lmo0818) was crystallized in an unknown functional state. The crystal belonged to space group P2(1)2(1)2(1) and a complete data set was collected to 3.2 Å resolution. The molecular-replacement solution obtained revealed that Lmo0818 is likely to adopt an E2-like state mimicking the phosphorylated intermediate in the functional cycle of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) and a stacked bilayer `type I' packing in the crystal.
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Purification, crystallization and preliminary crystallographic studies of a PacL homologue from Listeria monocytogenes.
By admin, on April 14th, 2012
J Biol Chem. 2012 Apr 12;
Okayasu M, Nakayachi M, Hayashida C, Ito J, Kaneda T, Masuhara M, Suda N, Sato T, Hakeda Y
Osteoporosis is associated with both atherosclerosis and vascular calcification attributed to hyperlipidemia; however, the cellular and molecular mechanisms explaining the parallel progression of these diseases remain unclear. Here, we used low-density lipoprotein receptor-knockout (LDLR-/-) mice to elucidate the role of LDLR in regulating the differentiation of osteoclasts, which are responsible for bone resorption. Culturing wild-type osteoclast precursors in medium containing LDL-depleted serum decreased receptor activator of NF-κB ligand (RANKL)-induced osteoclast formation, and this defect was additively rescued by simultaneous treatment with native and oxidized LDLs. Osteoclast precursors constitutively expressed LDLR in a RANKL-independent manner. Osteoclast formation from LDLR-/- osteoclast precursors was delayed, and the multinucleated cells formed in culture were smaller and contained fewer nuclei than wild-type cells, implying impaired cell-cell fusion. Despite these findings, RANK signaling, including the activation of Erk and Akt, was normal in LDLR-/- preosteoclasts, and RANKL-induced expression of NFATc1 (a master regulator of osteoclastogenesis), cathepsin K, and tartrate-resistant acid phosphatase was equivalent in LDLR-null and wild-type cells. In contrast, the amounts of the osteoclast fusion-related proteins v-ATPase V0 subunit d2 and dendritic cell-specific transmembrane protein in LDLR-/- plasma membranes were reduced when compared to wild-type, suggesting a correlation with impaired cell-cell fusion, which occurs on the plasma membrane. LDLR-/- mice consistently exhibited increased bone mass in vivo. This change was accompanied by decreases in bone resorption parameters, with no changes in bone formation parameters. These findings provide a novel mechanism for osteoclast differentiation and improve the understanding of the correlation between osteoclast formation and lipids.
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Low-density Lipoprotein Receptor Deficiency Causes Impaired Osteoclastogenesis and Increased Bone Mass in Mice Due to a Defect in Osteoclastic Cell-Cell Fusion.
By admin, on April 14th, 2012
J Biol Chem. 2012 Apr 11;
He P, Ng BG, Losfeld ME, Zhu W, Freeze HH
Many human inherited disorders cause protein N-glycosylation defects, but there are few cellular markers to test gene complementation for such defects. Plasma membrane glycoproteins are potential biomarkers because they may be reduced or even absent in plasma membranes of glycosylation-deficient cells. We combined stable isotope labeling by amino acids in cell culture (SILAC) with linear ion trap mass spectrometry (LTQ-Orbitrap) to identify and quantify membrane proteins from wild-type CHO and glycosylation deficient CHO (Lec9) cells. We identified 165 under-represented proteins from 1447 unique quantified proteins, including 18 N-glycosylated plasma membrane proteins. Using various methods we found that intercellular cell adhesion molecule 1 (ICAM-1) was reduced in Lec9 cells and in fibroblasts from 31 congenital disorder of glycosylation (CDG) patients compared to normal controls. Mannose supplementation of phosphomannose isomerase (PMI)-deficient CDG-Ib (MPI-CDG) cells and complementation with phosphormannomutase-2 (PMM2) in PMM2 deficient CDG-Ia (PMM2-CDG) cells partially corrected hypo-glycosylation based on increased ICAM-1 presence on the plasma membrane. These data indicate that ICAM-1 could be a useful hypo-glycosylation biomarker to assess gene complementation of CDG-I patient cells and monitor improved glycosylation in response to therapeutic drugs.
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Identification of intercellular cell adhesion molecule 1 (ICAM-1) as a hypo-glycosylation marker in congenital disorders of glycosylation cells.
By admin, on April 14th, 2012
J Biol Chem. 2012 Apr 11;
He P, Ng BG, Losfeld ME, Zhu W, Freeze HH
Many human inherited disorders cause protein N-glycosylation defects, but there are few cellular markers to test gene complementation for such defects. Plasma membrane glycoproteins are potential biomarkers because they may be reduced or even absent in plasma membranes of glycosylation-deficient cells. We combined stable isotope labeling by amino acids in cell culture (SILAC) with linear ion trap mass spectrometry (LTQ-Orbitrap) to identify and quantify membrane proteins from wild-type CHO and glycosylation deficient CHO (Lec9) cells. We identified 165 under-represented proteins from 1447 unique quantified proteins, including 18 N-glycosylated plasma membrane proteins. Using various methods we found that intercellular cell adhesion molecule 1 (ICAM-1) was reduced in Lec9 cells and in fibroblasts from 31 congenital disorder of glycosylation (CDG) patients compared to normal controls. Mannose supplementation of phosphomannose isomerase (PMI)-deficient CDG-Ib (MPI-CDG) cells and complementation with phosphormannomutase-2 (PMM2) in PMM2 deficient CDG-Ia (PMM2-CDG) cells partially corrected hypo-glycosylation based on increased ICAM-1 presence on the plasma membrane. These data indicate that ICAM-1 could be a useful hypo-glycosylation biomarker to assess gene complementation of CDG-I patient cells and monitor improved glycosylation in response to therapeutic drugs.
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Identification of intercellular cell adhesion molecule 1 (ICAM-1) as a hypo-glycosylation marker in congenital disorders of glycosylation cells.
By admin, on April 5th, 2012
J Biol Chem. 2012 Apr 3;
Singh AK, McMillan J, Bukiya AN, Burton B, Parrill AL, Dopico AM
Large conductance, Ca(2+)- and voltage-gated K(+) (BK) channel proteins are ubiquitously expressed in cell membranes and control a wide variety of biological processes. Membrane cholesterol regulates the activity of membrane-associated proteins, including BK channels. Cholesterol modulation of BK channels alters action potential firing, colonic ion transport, smooth muscle contractility, endothelial function, and the channel alcohol response. The structural bases underlying cholesterol-BK channel interaction are unknown. Such interaction is determined by strict chemical requirements for the sterol molecule, suggesting cholesterol recognition by a protein surface. Here, we demonstrate that cholesterol action on BK channel-forming cbv1 proteins is mediated by their cytosolic C-tail domain (CTD), where we identified seven cholesterol recognition/interaction amino acid consensus motifs (CRACs 4-10), a distinct feature of BK proteins. Cholesterol-sensitivity is provided by the membrane-adjacent CRAC4 where Val444, Tyr450 and Lys453 are required for cholesterol-sensing, with hydrogen bonding and hydrophobic interactions participating in cholesterol location and recognition. However, cumulative truncations or Tyr-to-Phe substitutions in CRACs 5-10 progressively blunt cholesterol-sensitivity, documenting involvement of multiple CRACs in cholesterol-BK channel interaction. In conclusion, our study provides for the first time the structural bases of BK channel cholesterol sensitivity; the presence of membrane-adjacent CRAC4 and the long CTD with several other CRAC motifs, which are not found in other members of the TM6 superfamily of ion channels, very likely explains the unique cholesterol sensitivity of BK channels.
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Multiple cholesterol recognition/interaction amino acid consensus (CRAC) motifs in the cytosolic C tail of the slo1 subunit determine cholesterol sensitivity of Ca2+- and voltage-gated K+ (BK) channels.
By admin, on April 5th, 2012
J Biol Chem. 2012 Apr 3;
Singh AK, McMillan J, Bukiya AN, Burton B, Parrill AL, Dopico AM
Large conductance, Ca(2+)- and voltage-gated K(+) (BK) channel proteins are ubiquitously expressed in cell membranes and control a wide variety of biological processes. Membrane cholesterol regulates the activity of membrane-associated proteins, including BK channels. Cholesterol modulation of BK channels alters action potential firing, colonic ion transport, smooth muscle contractility, endothelial function, and the channel alcohol response. The structural bases underlying cholesterol-BK channel interaction are unknown. Such interaction is determined by strict chemical requirements for the sterol molecule, suggesting cholesterol recognition by a protein surface. Here, we demonstrate that cholesterol action on BK channel-forming cbv1 proteins is mediated by their cytosolic C-tail domain (CTD), where we identified seven cholesterol recognition/interaction amino acid consensus motifs (CRACs 4-10), a distinct feature of BK proteins. Cholesterol-sensitivity is provided by the membrane-adjacent CRAC4 where Val444, Tyr450 and Lys453 are required for cholesterol-sensing, with hydrogen bonding and hydrophobic interactions participating in cholesterol location and recognition. However, cumulative truncations or Tyr-to-Phe substitutions in CRACs 5-10 progressively blunt cholesterol-sensitivity, documenting involvement of multiple CRACs in cholesterol-BK channel interaction. In conclusion, our study provides for the first time the structural bases of BK channel cholesterol sensitivity; the presence of membrane-adjacent CRAC4 and the long CTD with several other CRAC motifs, which are not found in other members of the TM6 superfamily of ion channels, very likely explains the unique cholesterol sensitivity of BK channels.
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Multiple cholesterol recognition/interaction amino acid consensus (CRAC) motifs in the cytosolic C tail of the slo1 subunit determine cholesterol sensitivity of Ca2+- and voltage-gated K+ (BK) channels.
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