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ABSTRACT: Apolipoprotein A-I (apoA-I) is the major protein component of high-density lipoprotein (HDL) and critical to maintenance of cholesterol homeostasis. During reverse cholesterol transport HDL transitions between an array of subclasses, differing in size and composition. This process requires apoA-I to adapt to changes in the shape of the HDL particle, transiting from an apo-lipoprotein to a myriad of HDL subclass specific conformations. Changes in apoA-I structure impart alterations in HDL-specific enzyme and receptor binding properties and thereby direct the HDL particle through the reverse cholesterol transport pathway. In this study, we used site directed spin label electron paramagnetic resonance (SDSL-EPR) spectroscopy to examine the conformational details of the apoA-I central domain on HDL. The motional dynamics and accessibility to hydrophobic/hydrophilic relaxation agents of apoA-I residues 99-163 on 9.6 nm rHDL was analyzed by EPR. In previous analyses we examined residues 6-98 and 164-238 (of apoA-I's 243 residue length) and combined with current results, we have generated a full-length map of rHDL-associated apoA-I's backbone structure. Remarkably, given that a majority of apoA-I's length is comprised of amphipathic helices, we identify non-helical residues, specifically the presence of a beta strand (residues 149-157). The significance of these non-helical residues is discussed along with the other features in the context of apoA-I function in contrast to recent models derived by other methods. This article is protected by copyright. All rights reserved.
FEBS Journal 05/2013; · 3.79 Impact Factor
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Jens O Lagerstedt,
Jitka Petrlova,
Silvia Hilt,
Antonin Marek,
Youngran Chung,
Renuka Sriram,
Madhu S Budamagunta,
Jean F Desreux,
David Thonon,
Thomas Jue,
Alex I Smirnov,
John C Voss
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ABSTRACT: We have engineered apolipoprotein A-I (apoA-I), a major protein constituent of high-density lipoprotein (HDL), to contain DOTA-chelated Gd(III) as an MRI contrast agent for the purpose of imaging reconstituted HDL (rHDL) biodistribution, metabolism and regulation in vivo. This protein contrast agent was obtained by attaching the thiol-reactive Gd[MTS-ADO3A] label at Cys residues replaced at four distinct positions (52, 55, 76 and 80) in apoA-I. MRI of infused mice previously showed that the Gd-labeled apoA-I migrates to both the liver and the kidney, the organs responsible for HDL catabolism; however, the contrast properties of apoA-I are superior when the ADO3A moiety is located at position 55, compared with the protein labeled at positions 52, 76 or 80. It is shown here that continuous wave X-band (9 GHz) electron paramagnetic resonance (EPR) spectroscopy is capable of detecting differences in the Gd(III) signal when comparing the labeled protein in the lipid-free with the rHDL state. Furthermore, the values of NMR relaxivity obtained for labeled variants in both the lipid-free and rHDL states correlate to the product of the X-band Gd(III) spectral width and the collision frequency between a nitroxide spin label and a polar relaxation agent. Consistent with its superior relaxivity measured by NMR, the rHDL-associated apoA-I containing the Gd[MTS-ADO3A] probe attached to position 55 displays favorable dynamic and water accessibility properties as determined by X-band EPR. While room temperature EPR requires >1 m m Gd(III)-labeled and only >10 µ m nitroxide-labeled protein to resolve the spectrum, the volume requirement is exceptionally low (~5 µl). Thus, X-band EPR provides a practical assessment for the suitability of imaging candidates containing the site-directed ADO3A contrast probe. Copyright © 2013 John Wiley & Sons, Ltd.
Contrast Media & Molecular Imaging 05/2013; 8(3):252-64. · 3.33 Impact Factor
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ABSTRACT: Lipid-free apoA-I and mature spherical HDL have been shown to induce glucose uptake in skeletal muscle. To exploit apoA-I and HDL states for diabetes therapy, further understanding of interaction between muscle and apoA-I is required. This study has examined if nascent discoidal HDL, in which apoA-I attains a different conformation from mature HDL and lipid-free states, could induce muscle glucose uptake and if a specific domain of apoA-I can mediate this effect. Using L6 myotubes stimulated with synthetic reconstituted discoidal HDL (rHDL), we show a glucose uptake effect comparable to insulin. Increased plasma membrane GLUT4 levels in ex vivo rHDL-stimulated myofibers from HA-GLUT4-GFP transgenic mice support this observation. rHDL increased phosphorylation of AMP kinase (AMPK) and acetyl-coA carboxylase (ACC) but not Akt. A survey of domain specific peptides of apoA-I showed that the lipid-free C-terminal 190-243 fragment increases plasma membrane GLUT4, promotes glucose uptake, and activates AMPK signaling but not Akt. This may be explained by changes in α-helical content of 190-243 fragment versus full-length lipid-free apoA-I as assessed by circular dichroism spectroscopy. JLR Discoidal HDL and the 190-243 peptide of apoA-I are potent agonists of glucose uptake in skeletal muscle and the C-terminal α-helical content of apoA-I may be an important determinant of this effect.
The Journal of Lipid Research 03/2013; · 5.56 Impact Factor
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ABSTRACT: The Gtr1 protein of Saccharomyces cerevisiae is a member of the RagA subfamily of the Ras-like small GTPase superfamily. Gtr1 has been implicated in various cellular processes. Particularly, the Switch regions in the GTPase domain of Gtr1 are essential for TORC1 activation and amino acid signaling. Therefore, knowledge about the biochemical activity of Gtr1 is required to understand its mode of action and regulation.
By employing tryptophan fluorescence analysis and radioactive GTPase assays, we demonstrate that Gtr1 can adopt two distinct GDP- and GTP-bound conformations, and that it hydrolyses GTP much slower than Ras proteins. Using cysteine mutagenesis of Arginine-37 and Valine-67, residues at the Switch I and II regions, respectively, we show altered GTPase activity and associated conformational changes as compared to the wild type protein and the cysteine-less mutant.
The extremely low intrinsic GTPase activity of Gtr1 implies requirement for interaction with activating proteins to support its physiological function. These findings as well as the altered properties obtained by mutagenesis in the Switch regions provide insights into the function of Gtr1 and its homologues in yeast and mammals.
BMC Biochemistry 06/2012; 13:11. · 1.99 Impact Factor
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ABSTRACT: Apolipoprotein A-I (apoA-I) is the major protein component of high density lipoproteins (HDL) and plays a central role in cholesterol metabolism. The lipid-free/lipid-poor form of apoA-I is the preferred substrate for the ATP-binding cassette transporter A1 (ABCA1). The interaction of apoA-I with ABCA1 leads to the formation of cholesterol laden high density lipoprotein (HDL) particles, a key step in reverse cholesterol transport and the maintenance of cholesterol homeostasis. Knowledge of the structure of lipid-free apoA-I is essential to understanding its critical interaction with ABCA1 and the molecular mechanisms underlying HDL biogenesis. We therefore examined the structure of lipid-free apoA-I by electron paramagnetic resonance spectroscopy (EPR). Through site directed spin label EPR, we mapped the secondary structure of apoA-I and identified sites of spin coupling as residues 26, 44, 64, 167, 217 and 226. We capitalize on the fact that lipid-free apoA-I self-associates in an anti-parallel manner in solution. We employed these sites of spin coupling to define the central plane in the dimeric apoA-I complex. Applying both the constraints of dipolar coupling with the EPR-derived pattern of solvent accessibility, we assembled the secondary structure into a tertiary context, providing a solution structure for lipid-free apoA-I. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).
Biochimica et Biophysica Acta 03/2012; 1821(3):448-55. · 4.66 Impact Factor
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ABSTRACT: Postprandial hyperlipemia, characterized by increased circulating very low-density lipoproteins (VLDL) and circulating lipopolysaccharide (LPS), has been proposed as a mechanism of vascular injury. Our goal was to examine the interactions between postprandial lipoproteins, LPS, and apoE3 and apoE4 on monocyte activation.
We showed that apoE3 complexed to phospholipid vesicles attenuates LPS-induced THP-1 monocyte cytokine expression, while apoE4 increases expression. ELISA revealed that apoE3 binds to LPS with higher affinity than apoE4. Electron paramagnetic resonance (EPR) spectroscopy of site-directed spin labels placed on specific amino acids of apoE3 showed that LPS interferes with conformational changes normally associated with lipid binding. Specifically, compared to apoE4, apoE bearing the E3-like R112→Ser mutation displays increased self association when exposed to LPS, consistent with a stronger apoE3-LPS interaction. Additionally, lipolysis of fasting VLDL from normal human donors attenuated LPS-induced TNFα secretion from monocytes to a greater extent than postprandial VLDL, an effect partially reversed by blocking apoE. This effect was reproduced using fasting VLDL lipolysis products from e3/e3 donors, but not from e4/e4 subjects, suggesting that apoE3 on fasting VLDL prevents LPS-induced inflammation more readily than apoE4.
Postprandial apoE isoform and conformational changes associated with VLDL dramatically modulate vascular inflammation.
PLoS ONE 01/2012; 7(11):e50513. · 4.09 Impact Factor
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ABSTRACT: A number of amyloidogenic variants of apoA-I have been discovered but most have not been analyzed. Previously, we showed that the G26R mutation of apoA-I leads to increased β-strand structure, increased N-terminal protease susceptibility, and increased fibril formation after several days of incubation. In vivo, this and other variants mutated in the N-terminal domain (residues 26 to ∼90) lead to renal and hepatic accumulation. In contrast, several mutations identified within residues 170 to 178 lead to cardiac, laryngeal, and cutaneous protein deposition. Here, we describe the structural changes in the fibrillogenic variant L178H. Like G26R, the initial structure of the protein exhibits altered tertiary conformation relative to wild-type protein along with decreased stability and an altered lipid binding profile. However, in contrast to G26R, L178H undergoes an increase in helical structure upon incubation at 37°C with a half time (t(1/2)) of about 12 days. Upon prolonged incubation, the L178H mutant forms fibrils of a diameter of 10 nm that ranges in length from 30 to 120 nm. These results show that apoA-I, known for its dynamic properties, has the ability to form multiple fibrillar conformations, which may play a role in the tissue-specific deposition of the individual variants.
The Journal of Lipid Research 12/2011; 53(3):390-8. · 5.56 Impact Factor
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Eva B. Nygaard, Jens O. Lagerstedt,
Gabriel Bjerre,
Biao Shi,
Madhu Budamagunta,
Kristian A. Poulsen,
Stine Meinild,
Robert R. Rigor,
John C. Voss,
Peter M. Cala,
Stine F. Pedersen
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ABSTRACT: We previously presented evidence that transmembrane domain (TM) IV and TM X-XI are important for inhibitor binding and ion
transport by the human Na+/H+ exchanger, hNHE1 (Pedersen, S. F., King, S. A., Nygaard, E. B., Rigor, R. R., and Cala, P. M. (2007) J. Biol. Chem. 282, 19716–19727). Here, we present a structural model of the transmembrane part of hNHE1 that further supports this conclusion.
The hNHE1 model was based on the crystal structure of the Escherichia coli Na+/H+ antiporter, NhaA, and previous cysteine scanning accessibility studies of hNHE1 and was validated by EPR spectroscopy of
spin labels in TM IV and TM XI, as well as by functional analysis of hNHE1 mutants. Removal of all endogenous cysteines in
hNHE1, introduction of the mutations A173C (TM IV) and/or I461C (TM XI), and expression of the constructs in mammalian cells
resulted in functional hNHE1 proteins. The distance between these spin labels was ∼15 A, confirming that TM IV and TM XI are
in close proximity. This distance was decreased both at pH 5.1 and in the presence of the NHE1 inhibitor cariporide. A similar
TM IV·TM XI distance and a similar change upon a pH shift were found for the cariporide-insensitive Pleuronectes americanus (pa) NHE1; however, in paNHE1, cariporide had no effect on TM IV·TM XI distance. The central role of the TM IV·TM XI arrangement
was confirmed by the partial loss of function upon mutation of Arg425, which the model predicts stabilizes this arrangement. The data are consistent with a role for TM IV and TM XI rearrangements
coincident with ion translocation and inhibitor binding by hNHE1.
Journal of Biological Chemistry 01/2011; 286(1):634-648. · 4.77 Impact Factor
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Renuka Sriram, Jens O Lagerstedt,
Jitka Petrlova,
Haris Samardzic,
Ulrike Kreutzer,
Hongtao Xie,
George A Kaysen,
Jean F Desreux,
David Thonon,
Vincent Jacques,
Martha Van Loan,
John C Rutledge,
Michael N Oda,
John C Voss,
Thomas Jue
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ABSTRACT: Coronary disease risk increases inversely with high-density lipoprotein (HDL) level. The measurement of the biodistribution and clearance of HDL in vivo, however, has posed a technical challenge. This study presents an approach to the development of a lipoprotein MRI agent by linking gadolinium methanethiosulfonate (Gd[MTS-ADO3A]) to a selective cysteine mutation in position 55 of apo AI, the major protein of HDL. The contrast agent targets both liver and kidney, the sites of HDL catabolism, whereas the standard MRI contrast agent, gadolinium-diethylenetriaminepentaacetic acid-bismethylamide (GdDTPA-BMA, gadodiamide), enhances only the kidney image. Using a modified apolipoprotein AI to create an HDL contrast agent provides a new approach to investigate HDL biodistribution, metabolism and regulation in vivo.
NMR in Biomedicine 01/2011; 24(7):916-24. · 3.21 Impact Factor
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ABSTRACT: Apolipoprotein A-I (apoA-I) is the major protein component of high density lipoproteins (HDL) and a critical element of cholesterol metabolism. To better elucidate the role of the apoA-I structure-function in cholesterol metabolism, the conformation of the apoA-I N terminus (residues 6-98) on nascent HDL was examined by electron paramagnetic resonance (EPR) spectroscopic analysis. A series of 93 apoA-I variants bearing single nitroxide spin label at positions 6-98 was reconstituted onto 9.6-nm HDL particles (rHDL). These particles were subjected to EPR spectral analysis, measuring regional flexibility and side chain solvent accessibility. Secondary structure was elucidated from side-chain mobility and molecular accessibility, wherein two major α-helical domains were localized to residues 6-34 and 50-98. We identified an unstructured segment (residues 35-39) and a β-strand (residues 40-49) between the two helices. Residues 14, 19, 34, 37, 41, and 58 were examined by EPR on 7.8, 8.4, and 9.6 nm rHDL to assess the effect of particle size on the N-terminal structure. Residues 14, 19, and 58 showed no significant rHDL size-dependent spectral or accessibility differences, whereas residues 34, 37, and 41 displayed moderate spectral changes along with substantial rHDL size-dependent differences in molecular accessibility. We have elucidated the secondary structure of the N-terminal domain of apoA-I on 9.6 nm rHDL (residues 6-98) and identified residues in this region that are affected by particle size. We conclude that the inter-helical segment (residues 35-49) plays a role in the adaptation of apoA-I to the particle size of HDL.
Journal of Biological Chemistry 11/2010; 286(4):2966-75. · 4.77 Impact Factor
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Eva B Nygaard, Jens O Lagerstedt,
Gabriel Bjerre,
Biao Shi,
Madhu Budamagunta,
Kristian A Poulsen,
Stine Meinild,
Robert R Rigor,
John C Voss,
Peter M Cala,
Stine F Pedersen
[show abstract]
[hide abstract]
ABSTRACT: We previously presented evidence that transmembrane domain (TM) IV and TM X-XI are important for inhibitor binding and ion transport by the human Na(+)/H(+) exchanger, hNHE1 (Pedersen, S. F., King, S. A., Nygaard, E. B., Rigor, R. R., and Cala, P. M. (2007) J. Biol. Chem. 282, 19716-19727). Here, we present a structural model of the transmembrane part of hNHE1 that further supports this conclusion. The hNHE1 model was based on the crystal structure of the Escherichia coli Na(+)/H(+) antiporter, NhaA, and previous cysteine scanning accessibility studies of hNHE1 and was validated by EPR spectroscopy of spin labels in TM IV and TM XI, as well as by functional analysis of hNHE1 mutants. Removal of all endogenous cysteines in hNHE1, introduction of the mutations A173C (TM IV) and/or I461C (TM XI), and expression of the constructs in mammalian cells resulted in functional hNHE1 proteins. The distance between these spin labels was ∼15 A, confirming that TM IV and TM XI are in close proximity. This distance was decreased both at pH 5.1 and in the presence of the NHE1 inhibitor cariporide. A similar TM IV·TM XI distance and a similar change upon a pH shift were found for the cariporide-insensitive Pleuronectes americanus (pa) NHE1; however, in paNHE1, cariporide had no effect on TM IV·TM XI distance. The central role of the TM IV·TM XI arrangement was confirmed by the partial loss of function upon mutation of Arg(425), which the model predicts stabilizes this arrangement. The data are consistent with a role for TM IV and TM XI rearrangements coincident with ion translocation and inhibitor binding by hNHE1.
Journal of Biological Chemistry 10/2010; 286(1):634-48. · 4.77 Impact Factor
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ABSTRACT: Maintaining normal physiological homeostasis depends upon a coordinated metabolism of both water-soluble and -insoluble substrates.
In humans the body derives these molecules — such as glucose, amino acids, and fatty acids — from complex food matter. Water-soluble
substrates can circulate readily in blood, while water-insoluble molecules — such as fatty acid, triacylglycerol, and cholesterol
— require ampiphathic carriers to transport them from the site of biosynthesis (liver and intestine) to the target tissue.
For fatty acid, albumin serves as the major transporter. For triacylglycerol and cholesterol, however, macromolecular complexes
aggregate the hydrophobic molecules into the core and cover the surface with amphiphatic proteins and phospholipids to solubilize
the particles in the lymphatic and circulatory systems. These macromolecules belong to a class of proteins, plasma lipoproteins,
with specific functions and cellular targets. In the clinic these lipoproteins prognosticate the risk of cardiovascular disease
(CVD). Lipoproteins divide usually into five major types: chylomicron, very-low-density lipoprotein (VLDL), intermediate-density
lipoprotein (IDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL). Each lipoprotein type exhibits characteristic
density, size, and composition. As implied in the name, the density varies from the low-density chylomicron (<0.95 g/ml) to
the high-density HDL (1.2 g/ml). Size also varies. The chylomicron has the largest diameter (75–1,200 nm), and HDL has the
smallest (5–12 nm). The physical property variation arises from each lipoprotein’s distinct composition. In a chylomicron,
cholesterol, triacylglycerol, and phospholipid predominate and constitute about 90% of the particle. Protein constitutes only
about 10%. In contrast, the smaller HDL has less cholesterol, triacylglycerol, and phospholipid (65% of the particle) but
more protein (over 30%).
06/2009: pages 285-297;
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ABSTRACT: In Saccharomyces cerevisiae, phosphate uptake is mainly dependent on the proton-coupled Pho84 permease under phosphate-limited growth conditions. Phosphate addition causes Pho84-mediated activation of the protein kinase A (PKA) pathway as well as rapid internalization and vacuolar breakdown of Pho84. We show that Pho84 undergoes phosphate-induced phosphorylation and subsequent ubiquitination on amino acids located in the large middle intracellular loop prior to endocytosis. The attachment of ubiquitin is dependent on the ubiquitin conjugating enzymes Ubc2 and Ubc4. In addition, we show that the Pho84 endocytotic process is delayed in strains with reduced PKA activity. Our results suggest that Pho84-mediated activation of the PKA pathway is responsible for its own downregulation by phosphorylation, ubiquination, internalization, and vacuolar breakdown.
Biochemistry 05/2009; 48(21):4497-505. · 3.42 Impact Factor
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ABSTRACT: We show that a two-step process, involving spontaneous self-assembly of lipids and apolipoproteins and surface patterning, produces single, supported lipid bilayers over two discrete and independently adjustable length scales. Specifically, an aqueous phase incubation of DMPC vesicles with purified apolipoprotein A-I results in the reconstitution of high density lipoprotein (rHDL), wherein nanoscale clusters of single lipid bilayers are corralled by the protein. Adsorption of these discoidal particles to clean hydrophilic glass (or silicon) followed by direct exposure to a spatial pattern of short-wavelength UV radiation directly produces microscopic patterns of nanostructured bilayers. Alternatively, simple incubation of aqueous phase rHDL with a chemically patterned hydrophilic/hydrophobic surface produces a novel compositional pattern, caused by an increased affinity for adsorption onto hydrophilic regions relative to the surrounding hydrophobic regions. Further, by simple chemical denaturation of the boundary protein, nanoscale compartmentalization can be selectively erased, thus producing patterns of laterally fluid, lipid bilayers structured solely at the mesoscopic length scale. Since these aqueous phase microarrays of nanostructured lipid bilayers allow for membrane proteins to be embedded within single nanoscale bilayer compartments, they present a viable means of generating high-density membrane protein arrays. Such a system would permit in-depth elucidation of membrane protein structure-function relationships and the consequences of membrane compartmentalization on lipid dynamics.
Journal of the American Chemical Society 08/2008; 130(33):11164-9. · 9.91 Impact Factor
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ABSTRACT: The single amino acid mutation G26R in human apolipoprotein A-I (apoA-IIOWA) leads to the formation of beta-secondary structure rich amyloid fibrils in vivo. Here we show that full-length apoA-IIOWA has a decreased lipid-binding capability, an increased amino-terminal sensitivity to protease, and a propensity to form annular protofibrils visible by electron microscopy. The molecular basis for the conversion of apolipoprotein A-I to a proamyloidogenic form was examined by electron paramagnetic resonance spectroscopy. Our recent findings [Lagerstedt, J. O., Budamagunta, M. S., Oda, M. N., and Voss, J. C. (2007) J. Biol. Chem. 282, 9143-9149] indicate that Gly26 in the native apoprotein separates a preceding beta-strand structure (residues 20-25) from a downstream largely alpha-helical region. The current study demonstrates that the G26R variant promotes a structural transition of positions 27-56 to a mixture of coil and beta-strand secondary structure. Microscopy and staining by amyloidophilic dyes suggest that this alteration extends throughout the protein within 1 week of incubation in vitro, leading to insoluble aggregates of distinct morphology. The severe consequences of the Iowa mutation likely arise from the combination of losing the contribution of the native Gly residue in terminating beta-strand propagation and the promotion of beta-structure when an Arg is introduced adjacent to the succeeding residue of identical charge and size, Arg27.
Biochemistry 09/2007; 46(34):9693-9. · 3.42 Impact Factor
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ABSTRACT: Apolipoprotein A-I (apoA-I) is the major protein constituent of high density lipoprotein (HDL) and plays a central role in phospholipid and cholesterol metabolism. This 243-residue long protein is remarkably flexible and assumes numerous lipid-dependent conformations. Consequently, definitive structural determination of lipid-free apoA-I in solution has been difficult. Using electron paramagnetic spectroscopy of site-directed spin labels in the N-terminal domain of apoA-I (residues 1-98) we have mapped a mixture of secondary structural elements, the composition of which is consistent with findings from other in-solution methods. Based on side chain mobility and their accessibility to polar and non-polar spin relaxers, the precise location of secondary elements for amino acids 14-98 was determined for both lipid-free and lipid-bound apoA-I. Based on intermolecular dipolar coupling at positions 26, 44, and 64, these secondary structural elements were arranged into a tertiary fold to generate a structural model for lipid-free apoA-I in solution.
Journal of Biological Chemistry 04/2007; 282(12):9143-9. · 4.77 Impact Factor
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Sophie Thuswaldner, Jens O Lagerstedt,
Marc Rojas-Stütz,
Karim Bouhidel,
Christophe Der,
Nathalie Leborgne-Castel,
Arti Mishra,
Francis Marty,
Benoît Schoefs,
Iwona Adamska,
Bengt L Persson,
Cornelia Spetea
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ABSTRACT: In plants the chloroplast thylakoid membrane is the site of light-dependent photosynthetic reactions coupled to ATP synthesis. The ability of the plant cell to build and alter this membrane system is essential for efficient photosynthesis. A nucleotide translocator homologous to the bovine mitochondrial ADP/ATP carrier (AAC) was previously found in spinach thylakoids. Here we have identified and characterized a thylakoid ATP/ADP carrier (TAAC) from Arabidopsis.(i) Sequence homology with the bovine AAC and the prediction of chloroplast transit peptides indicated a putative carrier encoded by the At5g01500 gene, as a TAAC. (ii) Transiently expressed TAAC-green fluorescent protein fusion construct was targeted to the chloroplast. Western blotting using a peptide-specific antibody together with immunogold electron microscopy revealed a major location of TAAC in the thylakoid membrane. Previous proteomic analyses identified this protein in chloroplast envelope preparations. (iii) Recombinant TAAC protein specifically imports ATP in exchange for ADP across the cytoplasmic membrane of Escherichia coli. Studies on isolated thylakoids from Arabidopsis confirmed these observations. (iv) The lack of TAAC in an Arabidopsis T-DNA insertion mutant caused a 30-40% reduction in the thylakoid ATP transport and metabolism. (v) TAAC is readily expressed in dark-grown Arabidopsis seedlings, and its level remains stable throughout the greening process. Its expression is highest in developing green tissues and in leaves undergoing senescence or abiotic stress. We propose that the TAAC protein supplies ATP for energy-dependent reactions during thylakoid biogenesis and turnover in plants.
Journal of Biological Chemistry 04/2007; 282(12):8848-59. · 4.77 Impact Factor
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Journal of Biological Chemistry. 01/2007;
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ABSTRACT: The Pho84 high-affinity phosphate permease is the primary phosphate transporter in the yeast Saccharomyces cerevisiae under phosphate-limiting conditions. The soluble G protein, Gtr1, has previously been suggested to be involved in the derepressible Pho84 phosphate uptake function. This idea was based on a displayed deletion phenotype of Deltagtr1 similar to the Deltapho84 phenotype. As of yet, the mode of interaction has not been described. The consequences of a deletion of gtr1 on in vivo Pho84 expression, trafficking and activity, and extracellular phosphatase activity were analyzed in strains synthesizing either Pho84-green fluorescent protein or Pho84-myc chimeras. The studies revealed a delayed response in Pho84-mediated phosphate uptake and extracellular phosphatase activity under phosphate-limiting conditions. EPR spectroscopic studies verified that the N-terminal G binding domain (residues 1-185) harbors the nucleotide responsive elements. In contrast, the spectra obtained for the C-terminal part (residues 186-310) displayed no evidence of conformational changes upon GTP addition.
Biochemistry 02/2005; 44(2):511-7. · 3.42 Impact Factor
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ABSTRACT: The phosphate transporter Pho84 of Saccharomyces cerevisiae is predicted to contain 12 transmembrane (TM) regions, divided into two partially duplicated parts of 6 TM segments. The three-dimensional (3D) organization of the Pho84 protein has not yet been determined. However, the 3D crystal structure of the Escherichia coli MFS glycerol-3-phosphate/phosphate antiporter, GlpT, and lactose transporter, LacY, has recently been determined. On the basis of extensive prediction and fold recognition analyses (at the MetaServer), GlpT was proposed as the best structural template on which the arrangement of TM segments of the Pho84 transporter was fit, using the comparative structural modeling program MODELLER. To initiate an evaluation of the appropriateness of the Pho84 model, we have performed two direct tests by targeting spin labels to putative TM segments 8 and 12. Electron paramagnetic resonance spectroscopy was then applied on purified and spin labeled Pho84. The line shape from labels located at both positions is consistent with the structural environment predicted by the template-generated model, thus supporting the model.
FEBS Letters 01/2005; 578(3):262-8. · 3.54 Impact Factor
