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Volkmar Braun - One of the best experts on this subject based on the ideXlab platform.
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Journal of Supramolecular Structure 5:37 (1 1)-58 (32) (1976) Functional Organization of the Outer Membrane of Escherichia Coli: Phage and Colicin Receptors as Components of Iron Uptake Systems
2013Co-Authors: Volkmar Braun, Klaus Hantke, Robert E. W. Hancock, Anton HartmannAbstract:The functional interaction of outer membrane proteins of E. coli can be studied using phage and colicin receptors which are essential components of penetration systems. The uptake of ferric iron in the form of the Ferrichrome complex requires the ton A and ton B functions in the outer membrane of E. coli. The ton A gene product is the receptor protein for phage T5 and is required together with the ton B function by the phages T1 and @SO to infect cells and by colicin M and the antibiotic albomycin, a structural analogue of Ferrichrome, to kill cells. The ton B function is necessary for the uptake of ferric iron complexed by citrate. Iron complexed by enterochelin is only transported in the presence of the ton B and feu functions. Cells which have lost the feu function are resistant to the colicins B, I or V while ton B mutants are resistant to all 3 colicins. The interaction of the ton A, ton B, and feu functions apparently permits quite different “substrates ” to overcome the permeability barrier of the outer membrane. It was shown for Ferrichrome dependent iron uptake that the complexing agent was not altered and could be used repeatedly. Only very low amounts of 3H-labeled Ferrichrome were found in the cell. It is possible that the iron is mobilized in the membrane and that desferriferrichrom
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the beta barrel domain of fhuadelta5 160 is sufficient for tonb dependent fhua activities of escherichia coli
Molecular Microbiology, 1999Co-Authors: Michael Braun, Helmut Killmann, Volkmar BraunAbstract:FhuA in the outer membrane of Escherichia coli serves as a transporter for Ferrichrome, the antibiotics albomycin and rifamycin CGP4832, colicin M, and as receptor for phages T1, T5 and φ80. The previously determined crystal structure reveals that residues 160–714 of the mature protein form a β-barrel that is closed from the periplasmic side by the globular N-proximal fragment, residues 1–159, designated the cork. In this study, deletion of the cork resulted in a stable protein, FhuAΔ5-160, that was incorporated in the outer membrane. Cells that synthesized FhuAΔ5-160 displayed a higher sensitivity to large antibiotics such as erythromycin, rifamycin, bacitracin and vancomycin, and grew on maltotetraose and maltopentaose in the absence of LamB. Higher concentrations of Ferrichrome supported growth of a tonB mutant that synthesized FhuAΔ5-160. These results demonstrate non-specific diffusion of compounds across the outer membrane of cells that synthesize FhuAΔ5-160. However, growth of a FhuAΔ5-160 tonB wild-type strain occurred at low Ferrichrome concentrations, and Ferrichrome was transported at about 45% of the FhuA wild-type rate despite the lack of Ferrichrome binding sites provided by the cork. FhuAΔ5-160 conferred sensitivity to the phages and colicin M at levels similar to that of wild-type FhuA, and to albomycin and rifamycin CGP 4832. The activity of FhuAΔ5-160 depended on TonB, although the mutant lacks the TonB box (residues 7–11) previously implicated in the interaction of FhuA with TonB. CCCP inhibited tonB-dependent transport of Ferrichrome through FhuAΔ5-160. FhuAΔ5-160 still functions as a specific transporter, and sites in addition to the TonB box are involved in the TonB-mediated response of FhuA to the proton gradient of the cytoplasmic membrane. It is proposed that TonB interacts with the TonB box of FhuA and with the β-barrel to release Ferrichrome from the FhuA binding sites and to open the channel in FhuA. For transport of Ferrichrome through the open channel of FhuAΔ5-160, interaction of TonB with the β-barrel is sufficient to release Ferrichrome from the residual binding sites at the β-barrel and to induce the active conformation of the L4 loop at the cell surface for infection by the TonB-dependent phages T1 and φ80.
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synthesis and activity of p azidobenzoyloxyferricrocin a photoactivatable analog of Ferrichrome
Biometals, 1999Co-Authors: Abdel Latif Salah El M Din, Volkmar Braun, Mohamed A AbdallahAbstract:p-azidobenzoyloxy desferriferricrocin (AF) 2, a photoactivatable analog of Ferrichrome, was prepared by selective acylation of the serine group of ferricrocin 1 in two steps: transesterification of ferricrocin followed by demetallation. A model compound, (L) 2-benzyloxycarbonylamino-3-p-azidobenzoyloxy N-isopropyl propionamide 8, was separately synthesized in order to set up optimal transesterification conditions to avoid α, β-elimination or epimerization of serine. Binding of iron-loaded AF (FeAF) to the FhuA outer membrane receptor protein of Escherichia coli AB2847 was demonstrated by inhibition of Ferrichrome transport, interference with the infection by the bacteriophage φ80 and with killing of cells by albomycin and colicin M. FeAF transported iron only weakly which indicates that the photoaffinity moiety is incompatible with transport or intracellular iron release from the siderophore.
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identification of a new site for Ferrichrome transport by comparison of the fhua proteins of escherichia coli salmonella paratyphi b salmonella typhimurium and pantoea agglomerans
Journal of Bacteriology, 1998Co-Authors: Helmut Killmann, Christina Herrmann, Helga Wolff, Volkmar BraunAbstract:ABSTRACT The fhuA genes of Salmonella paratyphi B,Salmonella typhimurium, and Pantoea agglomeranswere sequenced and compared with the known fhuA sequence ofEscherichia coli. The highly similar FhuA proteins displayed the largest difference in the predicted gating loop, which inE. coli controls the permeability of the FhuA channel and serves as the principal binding site for the phages T1, T5, and φ80. All the FhuA proteins contained the region in the gating loops required in E. coli for Ferrichrome and albomycin transport. The three subdomains required for phage binding were contained in the gating loop of S. paratyphi B which is infected by theE. coli phages, whereas two of the subdomains were deleted in S. typhimurium and P. agglomerans which are resistant to the E. coli phages. Small deletions in a surface loop adjacent to the gating loop, residues 236 to 243 and 236 to 248, inactivated E. coli FhuA with regard to transport of Ferrichrome and albomycin, but sensitivity to T1 and T5 was fully retained and sensitivity to φ80 and colicin M was reduced 10-fold. Full-size FhuA hybrid proteins of S. paratyphi B and S. typhimurium displayedS. paratyphi B FhuA activity when the hybrids contained two-thirds of either the N- or the C-terminal portions ofS. paratyphi B and displayed S. typhimurium FhuA activity to phage ES18 when the hybrid contained two-thirds of the N-terminal region of the S. typhimurium FhuA. The central segment of the S. paratyphi B FhuA flanked on both sides by S. typhimurium FhuA regions conferred full sensitivity only to phage T5. The data support the essential role of the gating loop for the transport of Ferrichrome and albomycin, identified an additional loop for Ferrichrome and albomycin uptake, and suggest that several segments and their proper conformation, determined by the entire FhuA protein, contribute to the multiple FhuA activities.
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Conversion of the coprogen transport protein FhuE and the ferrioxamine B transport protein FoxA into Ferrichrome transport proteins
FEMS microbiology letters, 1998Co-Authors: Helmut Killmann, Volkmar BraunAbstract:The FhuA protein of Escherichia coli K-12 transports Ferrichrome and the structurally related antibiotic albomycin across the outer membrane and serves as a receptor for the phages T1, T5, and φ80 and for colicin M. In this paper, we show that chimeric proteins consisting of the central part of FhuA and the N- and C-terminal parts of FhuE (coprogen receptor) or the N- and/or C-terminal parts of FoxA (ferrioxamine B receptor), function as Ferrichrome transport proteins. Although the hybrid proteins contained the previously identified gating loop of FhuA, which is the principal binding site of the phages T5, T1, and φ80, only the hybrid protein consisting of the N-terminal third of FoxA and the C-terminal two thirds of FhuA conferred weak phage sensitivity to cells. Apparently, the gating loop is essential, but not sufficient for wild-type levels of Ferrichrome transport and for phage sensitivity. The properties of FhuA-FoxA hybrids suggest different regions of the two receptors for ferric siderophore uptake.
Caroline C Philpott - One of the best experts on this subject based on the ideXlab platform.
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phosphatidylserine is involved in the Ferrichrome induced plasma membrane trafficking of arn1 in saccharomyces cerevisiae
Journal of Biological Chemistry, 2010Co-Authors: Yan Guo, Minoo Shakouryelizeh, Olga Protchenko, Munira A Basrai, William A Prinz, Caroline C PhilpottAbstract:Arn1 is an integral membrane protein that mediates the uptake of Ferrichrome, an important nutritional source of iron, in Saccharomyces cerevisiae. In the absence of Ferrichrome, Arn1p is sorted directly from the trans-Golgi network to the vacuolar lumen for degradation. In the presence of low levels of Ferrichrome, the siderophore binds to a receptor domain on Arn1, triggering the redistribution of Arn1 to the plasma membrane. When extracellular Ferrichrome levels are high, Arn1 cycles between the plasma membrane and intracellular vesicles. To further understand the mechanisms of trafficking of Arn1p, we screened 4580 viable yeast deletion mutants for mislocalization of Arn1-GFP using synthetic genetic array technology. We identified over 100 genes required for trans-Golgi network-to-vacuole trafficking of Arn1-GFP and only two genes, SER1 and SER2, required for the Ferrichrome-induced plasma membrane trafficking of Arn1-GFP. SER1 and SER2 encode two enzymes of the major serine biosynthetic pathway, and the Arn1 trafficking defect in the ser1Δ strain was corrected with supplemental serine or glycine. Plasma membrane trafficking of Hxt3, a structurally related glucose transporter, was unaffected by SER1 deletion. Serine is required for the synthesis of multiple cellular components, including purines, sphingolipids, and phospholipids, but of these only phosphatidylserine corrected the Arn1 trafficking defects of the ser1Δ strain. Strains with defects in phospholipid synthesis also exhibited alterations in Arn1p trafficking, indicating that the intracellular trafficking of some transporters is dependent on the phospholipid composition of the cellular membranes.
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gga2 and ubiquitin dependent trafficking of arn1 the Ferrichrome transporter of saccharomyces cerevisiae
Molecular Biology of the Cell, 2007Co-Authors: Youngwoo Kim, Yi Deng, Caroline C PhilpottAbstract:The intracellular trafficking of Arn1, a Ferrichrome transporter in Saccharomyces cerevisiae, is controlled in part by the binding of Ferrichrome to the transporter. In the absence of Ferrichrome, Arn1 is sorted directly from the Golgi to endosomes. Ferrichrome binding triggers the redistribution of Arn1 to the plasma membrane, whereas Ferrichrome transport is associated with the cycling of Arn1 between the plasma membrane and endosomes. Here, we report that the clathrin adaptor Gga2 and ubiquitination by the Rsp5 ubiquitin ligase are required for trafficking of Arn1. Gga2 was required for Golgi-to-endosomal trafficking of Arn1, which was sorted from endosomes to the vacuole for degradation. Trafficking into the vacuolar lumen was dependent on ubiquitination by Rsp5, but ubiquitination was not required for plasma membrane accumulation of Arn1 in the presence of Ferrichrome. Retrograde trafficking via the retromer complex or Snx4 was also not required for plasma membrane accumulation. High concentrations of Ferrichrome led to higher levels of ubiquitination of Arn1, but they did not induce degradation. Without this ubiquitination, Arn1 remained on the plasma membrane, where it was active for transport. Arn1 was preferentially modified with polyubiquitin chains on a cluster of lysine residues at the amino terminus of the transporter.
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a receptor domain controls the intracellular sorting of the Ferrichrome transporter arn1
The EMBO Journal, 2005Co-Authors: Youngwoo Kim, Sarah M Lampert, Caroline C PhilpottAbstract:The Saccharomyces cerevisiae transporter Arn1p takes up the ferric-siderophore Ferrichrome, and extracellular Ferrichrome dramatically influences the intracellular trafficking of Arn1p. In the absence of Ferrichrome, Arn1p sorts directly to the endosomal compartment. At low concentrations of Ferrichrome, Arn1p stably relocalizes to the plasma membrane, yet little to no uptake of Ferrichrome occurs at these low concentrations. At higher concentrations of Ferrichrome, Arn1p cycles between the plasma membrane and endosome. Arn1p contains two binding sites for Ferrichrome: one site has an affinity similar to the KT for transport, but the second site has a much higher affinity. Here we report that this high-affinity binding site lies within a unique extracytosolic, carboxyl-terminal domain. Mutations within this domain lead to loss of Ferrichrome binding and uptake activities and missorting of Arn1p, including a failure to relocalize to the plasma membrane in the presence of Ferrichrome. Mutation of phenylalanine residues in the cytosolic tail of Arn1p also lead to missorting, but without defects in Ferrichrome binding. We propose that the carboxyl terminus of Arn1p contains a receptor domain that controls the intracellular trafficking of the transporter.
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the mechanism of Ferrichrome transport through arn1p and its metabolism in saccharomyces cerevisiae
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Robert E Moore, Youngwoo Kim, Caroline C PhilpottAbstract:Siderophores are low molecular weight compounds, synthesized and secreted by microorganisms, that specifically bind ferric iron with exceptionally high affinity. Microbes capture these compounds and take up the bound iron through specific, high-affinity systems. Saccharomyces cerevisiae can take up iron bound to siderophores through the transporters of the ARN family; however, the mechanism by which the siderophore-bound iron enters the cell via these transporters is not known. Here we describe how Ferrichrome, a siderophore of the hydroxamate class, is taken up by Arn1p. Arn1p exhibits two surface binding sites for Ferrichrome, one that is similar in affinity to the KT for uptake and one of a much higher affinity that is specific for the metallated form of Ferrichrome. Ferrichrome may gain access to the higher-affinity site through endocytosis. Tracer studies using 14C-labeled Ferrichrome bound to either iron(III) or aluminum(III), a nonreducible ligand for Ferrichrome, indicate that Ferrichrome enters the cell as the intact metallosiderophore and accumulates in the cytosol. Both Ferrichrome chelates were relatively stable within the cell, and metal-free Ferrichrome did not accumulate, indicating a role for Ferrichrome in intracellular iron storage. Iron stored as Ferrichrome was readily mobilized to meet the metabolic needs of the cell.
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Ferrichrome induces endosome to plasma membrane cycling of the Ferrichrome transporter arn1p in saccharomyces cerevisiae
The EMBO Journal, 2002Co-Authors: Youngwoo Kim, Cheol Won Yun, Caroline C PhilpottAbstract:Siderophores are small iron-binding molecules that are synthesized and secreted in the iron-free form by microorganisms. Saccharomyces cerevisiae takes up iron bound to siderophores by two separate systems, one of which requires the ARN family of sidero phore–iron transporters. Arn1p and Arn3p are expressed in endosome-like intracellular vesicles. Here we present evidence that, in the absence of its specific substrate, Ferrichrome, Arn1p is sorted directly from the Golgi to the endosomal compartment and does not cycle to the plasma membrane. When cells are exposed to Ferrichrome at low concentrations, Arn1p stably relocalizes to the plasma membrane. At higher concentrations of Ferrichrome, Arn1p relocalizes to the plasma membrane and rapidly undergoes endocytosis. Plasma membrane localization of Arn1p occurs only in the presence of its specific substrate, and not in the presence of other siderophores. Despite expression of Arn1p on the plasma membrane, mutant strains with defects in endocytosis exhibit reduced uptake of Ferrichrome–iron. Thus, siderophores influence the trafficking of the Arn transporters within the cell and this trafficking is important for transporter function.
Helmut Killmann - One of the best experts on this subject based on the ideXlab platform.
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determination of Ferrichrome binding to the fhua outer membrane transport protein periplasmic accumulation of Ferrichrome or transport of Ferrichrome into cells using a three layer oil technique
Analytical Biochemistry, 2002Co-Authors: Helmut Killmann, Glikeria GestwaAbstract:A new method for the determination of Ferrichrome binding to the FhuA transporter in the Escherichia coli outer membrane, Ferrichrome accumulation in the periplasmic space, and Ferrichrome transport into the cytoplasm was developed. Cells were separated from residual, soluble, radiolabeled Ferrichrome by centrifugation in a micro-test tube containing three layers of nonmixable solutions of different densities. Cells in the upper aqueous layer passed through the middle silicone oil layer, but did not enter the underlying NaI layer, thereby accumulating on top of the NaI layer; soluble compounds remained in the upper aqueous layer. Cells were then easily recovered by centrifugation, and radioactivity was determined by liquid scintillation counting. Reproducible results for all applications tested were obtained without the need for any washing steps. The method was tested by determination of receptor binding and transport of Ferrichrome with various FhuA mutants which, in contrast to their transport activity, showed only a weak binding of Ferrichrome to FhuA and compared with the commonly used cellulose nitrate filter method. Similar transport rates were obtained with the two methods, but binding of Ferrichrome to the mutated FhuA proteins and accumulation of Ferrichrome in the periplasm could be measured only with the new method.
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the beta barrel domain of fhuadelta5 160 is sufficient for tonb dependent fhua activities of escherichia coli
Molecular Microbiology, 1999Co-Authors: Michael Braun, Helmut Killmann, Volkmar BraunAbstract:FhuA in the outer membrane of Escherichia coli serves as a transporter for Ferrichrome, the antibiotics albomycin and rifamycin CGP4832, colicin M, and as receptor for phages T1, T5 and φ80. The previously determined crystal structure reveals that residues 160–714 of the mature protein form a β-barrel that is closed from the periplasmic side by the globular N-proximal fragment, residues 1–159, designated the cork. In this study, deletion of the cork resulted in a stable protein, FhuAΔ5-160, that was incorporated in the outer membrane. Cells that synthesized FhuAΔ5-160 displayed a higher sensitivity to large antibiotics such as erythromycin, rifamycin, bacitracin and vancomycin, and grew on maltotetraose and maltopentaose in the absence of LamB. Higher concentrations of Ferrichrome supported growth of a tonB mutant that synthesized FhuAΔ5-160. These results demonstrate non-specific diffusion of compounds across the outer membrane of cells that synthesize FhuAΔ5-160. However, growth of a FhuAΔ5-160 tonB wild-type strain occurred at low Ferrichrome concentrations, and Ferrichrome was transported at about 45% of the FhuA wild-type rate despite the lack of Ferrichrome binding sites provided by the cork. FhuAΔ5-160 conferred sensitivity to the phages and colicin M at levels similar to that of wild-type FhuA, and to albomycin and rifamycin CGP 4832. The activity of FhuAΔ5-160 depended on TonB, although the mutant lacks the TonB box (residues 7–11) previously implicated in the interaction of FhuA with TonB. CCCP inhibited tonB-dependent transport of Ferrichrome through FhuAΔ5-160. FhuAΔ5-160 still functions as a specific transporter, and sites in addition to the TonB box are involved in the TonB-mediated response of FhuA to the proton gradient of the cytoplasmic membrane. It is proposed that TonB interacts with the TonB box of FhuA and with the β-barrel to release Ferrichrome from the FhuA binding sites and to open the channel in FhuA. For transport of Ferrichrome through the open channel of FhuAΔ5-160, interaction of TonB with the β-barrel is sufficient to release Ferrichrome from the residual binding sites at the β-barrel and to induce the active conformation of the L4 loop at the cell surface for infection by the TonB-dependent phages T1 and φ80.
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identification of a new site for Ferrichrome transport by comparison of the fhua proteins of escherichia coli salmonella paratyphi b salmonella typhimurium and pantoea agglomerans
Journal of Bacteriology, 1998Co-Authors: Helmut Killmann, Christina Herrmann, Helga Wolff, Volkmar BraunAbstract:ABSTRACT The fhuA genes of Salmonella paratyphi B,Salmonella typhimurium, and Pantoea agglomeranswere sequenced and compared with the known fhuA sequence ofEscherichia coli. The highly similar FhuA proteins displayed the largest difference in the predicted gating loop, which inE. coli controls the permeability of the FhuA channel and serves as the principal binding site for the phages T1, T5, and φ80. All the FhuA proteins contained the region in the gating loops required in E. coli for Ferrichrome and albomycin transport. The three subdomains required for phage binding were contained in the gating loop of S. paratyphi B which is infected by theE. coli phages, whereas two of the subdomains were deleted in S. typhimurium and P. agglomerans which are resistant to the E. coli phages. Small deletions in a surface loop adjacent to the gating loop, residues 236 to 243 and 236 to 248, inactivated E. coli FhuA with regard to transport of Ferrichrome and albomycin, but sensitivity to T1 and T5 was fully retained and sensitivity to φ80 and colicin M was reduced 10-fold. Full-size FhuA hybrid proteins of S. paratyphi B and S. typhimurium displayedS. paratyphi B FhuA activity when the hybrids contained two-thirds of either the N- or the C-terminal portions ofS. paratyphi B and displayed S. typhimurium FhuA activity to phage ES18 when the hybrid contained two-thirds of the N-terminal region of the S. typhimurium FhuA. The central segment of the S. paratyphi B FhuA flanked on both sides by S. typhimurium FhuA regions conferred full sensitivity only to phage T5. The data support the essential role of the gating loop for the transport of Ferrichrome and albomycin, identified an additional loop for Ferrichrome and albomycin uptake, and suggest that several segments and their proper conformation, determined by the entire FhuA protein, contribute to the multiple FhuA activities.
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Conversion of the coprogen transport protein FhuE and the ferrioxamine B transport protein FoxA into Ferrichrome transport proteins
FEMS microbiology letters, 1998Co-Authors: Helmut Killmann, Volkmar BraunAbstract:The FhuA protein of Escherichia coli K-12 transports Ferrichrome and the structurally related antibiotic albomycin across the outer membrane and serves as a receptor for the phages T1, T5, and φ80 and for colicin M. In this paper, we show that chimeric proteins consisting of the central part of FhuA and the N- and C-terminal parts of FhuE (coprogen receptor) or the N- and/or C-terminal parts of FoxA (ferrioxamine B receptor), function as Ferrichrome transport proteins. Although the hybrid proteins contained the previously identified gating loop of FhuA, which is the principal binding site of the phages T5, T1, and φ80, only the hybrid protein consisting of the N-terminal third of FoxA and the C-terminal two thirds of FhuA conferred weak phage sensitivity to cells. Apparently, the gating loop is essential, but not sufficient for wild-type levels of Ferrichrome transport and for phage sensitivity. The properties of FhuA-FoxA hybrids suggest different regions of the two receptors for ferric siderophore uptake.
Youngwoo Kim - One of the best experts on this subject based on the ideXlab platform.
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gga2 and ubiquitin dependent trafficking of arn1 the Ferrichrome transporter of saccharomyces cerevisiae
Molecular Biology of the Cell, 2007Co-Authors: Youngwoo Kim, Yi Deng, Caroline C PhilpottAbstract:The intracellular trafficking of Arn1, a Ferrichrome transporter in Saccharomyces cerevisiae, is controlled in part by the binding of Ferrichrome to the transporter. In the absence of Ferrichrome, Arn1 is sorted directly from the Golgi to endosomes. Ferrichrome binding triggers the redistribution of Arn1 to the plasma membrane, whereas Ferrichrome transport is associated with the cycling of Arn1 between the plasma membrane and endosomes. Here, we report that the clathrin adaptor Gga2 and ubiquitination by the Rsp5 ubiquitin ligase are required for trafficking of Arn1. Gga2 was required for Golgi-to-endosomal trafficking of Arn1, which was sorted from endosomes to the vacuole for degradation. Trafficking into the vacuolar lumen was dependent on ubiquitination by Rsp5, but ubiquitination was not required for plasma membrane accumulation of Arn1 in the presence of Ferrichrome. Retrograde trafficking via the retromer complex or Snx4 was also not required for plasma membrane accumulation. High concentrations of Ferrichrome led to higher levels of ubiquitination of Arn1, but they did not induce degradation. Without this ubiquitination, Arn1 remained on the plasma membrane, where it was active for transport. Arn1 was preferentially modified with polyubiquitin chains on a cluster of lysine residues at the amino terminus of the transporter.
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a receptor domain controls the intracellular sorting of the Ferrichrome transporter arn1
The EMBO Journal, 2005Co-Authors: Youngwoo Kim, Sarah M Lampert, Caroline C PhilpottAbstract:The Saccharomyces cerevisiae transporter Arn1p takes up the ferric-siderophore Ferrichrome, and extracellular Ferrichrome dramatically influences the intracellular trafficking of Arn1p. In the absence of Ferrichrome, Arn1p sorts directly to the endosomal compartment. At low concentrations of Ferrichrome, Arn1p stably relocalizes to the plasma membrane, yet little to no uptake of Ferrichrome occurs at these low concentrations. At higher concentrations of Ferrichrome, Arn1p cycles between the plasma membrane and endosome. Arn1p contains two binding sites for Ferrichrome: one site has an affinity similar to the KT for transport, but the second site has a much higher affinity. Here we report that this high-affinity binding site lies within a unique extracytosolic, carboxyl-terminal domain. Mutations within this domain lead to loss of Ferrichrome binding and uptake activities and missorting of Arn1p, including a failure to relocalize to the plasma membrane in the presence of Ferrichrome. Mutation of phenylalanine residues in the cytosolic tail of Arn1p also lead to missorting, but without defects in Ferrichrome binding. We propose that the carboxyl terminus of Arn1p contains a receptor domain that controls the intracellular trafficking of the transporter.
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the mechanism of Ferrichrome transport through arn1p and its metabolism in saccharomyces cerevisiae
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Robert E Moore, Youngwoo Kim, Caroline C PhilpottAbstract:Siderophores are low molecular weight compounds, synthesized and secreted by microorganisms, that specifically bind ferric iron with exceptionally high affinity. Microbes capture these compounds and take up the bound iron through specific, high-affinity systems. Saccharomyces cerevisiae can take up iron bound to siderophores through the transporters of the ARN family; however, the mechanism by which the siderophore-bound iron enters the cell via these transporters is not known. Here we describe how Ferrichrome, a siderophore of the hydroxamate class, is taken up by Arn1p. Arn1p exhibits two surface binding sites for Ferrichrome, one that is similar in affinity to the KT for uptake and one of a much higher affinity that is specific for the metallated form of Ferrichrome. Ferrichrome may gain access to the higher-affinity site through endocytosis. Tracer studies using 14C-labeled Ferrichrome bound to either iron(III) or aluminum(III), a nonreducible ligand for Ferrichrome, indicate that Ferrichrome enters the cell as the intact metallosiderophore and accumulates in the cytosol. Both Ferrichrome chelates were relatively stable within the cell, and metal-free Ferrichrome did not accumulate, indicating a role for Ferrichrome in intracellular iron storage. Iron stored as Ferrichrome was readily mobilized to meet the metabolic needs of the cell.
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Ferrichrome induces endosome to plasma membrane cycling of the Ferrichrome transporter arn1p in saccharomyces cerevisiae
The EMBO Journal, 2002Co-Authors: Youngwoo Kim, Cheol Won Yun, Caroline C PhilpottAbstract:Siderophores are small iron-binding molecules that are synthesized and secreted in the iron-free form by microorganisms. Saccharomyces cerevisiae takes up iron bound to siderophores by two separate systems, one of which requires the ARN family of sidero phore–iron transporters. Arn1p and Arn3p are expressed in endosome-like intracellular vesicles. Here we present evidence that, in the absence of its specific substrate, Ferrichrome, Arn1p is sorted directly from the Golgi to the endosomal compartment and does not cycle to the plasma membrane. When cells are exposed to Ferrichrome at low concentrations, Arn1p stably relocalizes to the plasma membrane. At higher concentrations of Ferrichrome, Arn1p relocalizes to the plasma membrane and rapidly undergoes endocytosis. Plasma membrane localization of Arn1p occurs only in the presence of its specific substrate, and not in the presence of other siderophores. Despite expression of Arn1p on the plasma membrane, mutant strains with defects in endocytosis exhibit reduced uptake of Ferrichrome–iron. Thus, siderophores influence the trafficking of the Arn transporters within the cell and this trafficking is important for transporter function.
Cheol Won Yun - One of the best experts on this subject based on the ideXlab platform.
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Ferrichrome induces endosome to plasma membrane cycling of the Ferrichrome transporter arn1p in saccharomyces cerevisiae
The EMBO Journal, 2002Co-Authors: Youngwoo Kim, Cheol Won Yun, Caroline C PhilpottAbstract:Siderophores are small iron-binding molecules that are synthesized and secreted in the iron-free form by microorganisms. Saccharomyces cerevisiae takes up iron bound to siderophores by two separate systems, one of which requires the ARN family of sidero phore–iron transporters. Arn1p and Arn3p are expressed in endosome-like intracellular vesicles. Here we present evidence that, in the absence of its specific substrate, Ferrichrome, Arn1p is sorted directly from the Golgi to the endosomal compartment and does not cycle to the plasma membrane. When cells are exposed to Ferrichrome at low concentrations, Arn1p stably relocalizes to the plasma membrane. At higher concentrations of Ferrichrome, Arn1p relocalizes to the plasma membrane and rapidly undergoes endocytosis. Plasma membrane localization of Arn1p occurs only in the presence of its specific substrate, and not in the presence of other siderophores. Despite expression of Arn1p on the plasma membrane, mutant strains with defects in endocytosis exhibit reduced uptake of Ferrichrome–iron. Thus, siderophores influence the trafficking of the Arn transporters within the cell and this trafficking is important for transporter function.
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siderophore iron uptake in saccharomyces cerevisiae identification of Ferrichrome and fusarinine transporters
Journal of Biological Chemistry, 2000Co-Authors: Cheol Won Yun, Robert E Moore, John Tiedeman, Caroline C PhilpottAbstract:A family of four putative transporters (Arn1p–4p) in Saccharomyces cerevisiae is expressed under conditions of iron deprivation and is regulated by Aft1p, the major iron-dependent transcription factor in yeast. One of these, Arn3p/Sit1p, facilitates the uptake of ferrioxamine B, a siderophore of the hydroxamate class. Here we report that ARN family members facilitated the uptake of iron from the trihydroxamate siderophores Ferrichrome, Ferrichrome A, and triacetylfusarinine C. Uptake of siderophore-bound iron was dependent on either the high-affinity ferrous iron transport system or the ARNfamily of transporters. The specificity of each siderophore for individual transporters was determined. Uptake of Ferrichrome and Ferrichrome A was facilitated by both Arn1p and Arn3p. Uptake of triacetylfusarinine C was facilitated by Arn2p, although small amounts of uptake also occurred through Arn1p and Arn3p. In contrast to the trihydroxamates, uptake of iron from the dihydroxamate rhodotorulic acid occurred only via the high-affinity ferrous iron system. Epitope-tagged Arn1p was expressed in intracellular vesicles in a pattern that was indistinguishable from that of Arn3p, whereas Ftr1p, a component of the high-affinity ferrous system, was expressed on the plasma membrane. These data indicate that S. cerevisiaemaintains two systems of siderophore uptake, only one of which is located on the plasma membrane.