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Sökning: WFRF:(Lagerstedt Jens O.)

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  • Lagerstedt, Jens O., 1975, et al. (författare)
  • Structural modeling of dual-affinity purified Pho84 phosphate transporter
  • 2004
  • Ingår i: FEBS Lett. - : Wiley-Blackwell. - 0014-5793 .- 1873-3468. ; 578:3, s. 262-8
  • Tidskriftsartikel (refereegranskat)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.
  • Sengottaiyan, Palanivelu, et al. (författare)
  • The intrinsic GTPase activity of the Gtr1 protein from Saccharomyces cerevisiae
  • 2012
  • Ingår i: BMC Biochemistry. - : BioMed Central (BMC). - 1471-2091. ; 13
  • Tidskriftsartikel (refereegranskat)abstract
    • Background: 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 [R. Gong, L. Li, Y. Liu, P. Wang, H. Yang, L. Wang, J. Cheng, K. L. Guan, Y. Xu, Genes Dev. 25 (2011) 1668-1673]. Therefore, knowledge about the biochemical activity of Gtr1 is required to understand its mode of action and regulation. Results: 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. Conclusions: 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.
  • Lagerstedt, Jens O., 1975, et al. (författare)
  • Electron paramagnetic resonance spectroscopy of site-directed spin labels reveals the structural heterogeneity in the N-terminal domain of apoA-I in solution
  • 2007
  • Ingår i: J Biol Chem. - : ASBMB. - 0021-9258. ; 282:12, s. 9143-9
  • Tidskriftsartikel (refereegranskat)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.
  • Lagerstedt, Jens O., 1975, et al. (författare)
  • Mapping the structural transition in an amyloidogenic apolipoprotein A-I
  • 2007
  • Ingår i: Biochemistry. - : The American Chemical Society (ACS). - 0006-2960. ; 46:34, s. 9693-9
  • Tidskriftsartikel (refereegranskat)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.
  • Lagerstedt, Jens O., 1975, et al. (författare)
  • Structure and function of the GTP binding protein Gtr1 and its role in phosphate transport in Saccharomyces cerevisiae
  • 2005
  • Ingår i: Biochemistry. - : The American Chemical Society (ACS). - 0006-2960. ; 44:2, s. 511-7
  • Tidskriftsartikel (refereegranskat)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.
  • Pratt, J. R., et al. (författare)
  • Effects of methylphosphonate, a phosphate analogue, on the expression and degradation of the high-affinity phosphate transporter Pho84, in Saccharomyces cerevisiae
  • 2004
  • Ingår i: Biochemistry. - : The American Chemical Society (ACS). - 0006-2960. ; 43:45, s. 14444-53
  • Tidskriftsartikel (refereegranskat)abstract
    • In Saccharomyces cerevisiae, the Pho84 high-affinity transport system is the major phosphate transporter activated when the cells experience a limitation in external phosphate. In this study, we have compared the phosphate-responsive mechanism of cells expressing PHO84 with a Deltapho84 strain by use of a phosphate analogue, methylphosphonate, which was judged to be suitable for assessment of phosphate homeostasis in the cells. Intracellular levels of the analogue, which in several respects mimicks phosphate, were monitored by (31)P NMR spectroscopy. Results show that methylphosphonate is a nonhydrolyzable and nonutilizable analogue that cannot be used to replenish phosphate or polyphosphate in yeast cells grown under conditions of phosphate limitation. However, the presence of methylphosphonate under such conditions represses the Pho5 acidic phosphatase activity of PHO84 cells, a finding that implies a direct role of the analogue in the regulation of phosphate-responsive genes and/or proteins. Likewise, accumulation of the Pho84 protein at the plasma membrane of the same cells is inhibited by methylphosphonate, although the derepressive expression of the PHO84 gene is unperturbed. Thus, a post-transcriptional regulation is suggested. Supportive of this suggestion is the fact that addition of methylphosphonate to cells with abundant and active Pho84 at the plasma membrane causes enhanced internalization of the Pho84 protein. Altogether, these observations suggest that the Pho84 transporter is regulated not only at the transcriptional level but also by a direct molecule-sensing mechanism at the protein level.
  • Thuswaldner, S., et al. (författare)
  • Identification, expression, and functional analyses of a thylakoid ATP/ADP carrier from Arabidopsis
  • 2007
  • Ingår i: J Biol Chem. - : ASBMB. - 0021-9258 .- 1083-351X. ; 282:12, s. 8848-59
  • Tidskriftsartikel (refereegranskat)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.
  • Lagerstedt, Jens O., et al. (författare)
  • Mutagenic and functional analysis of the C-terminus of Saccharomyces cerevisiae Pho84 phosphate transporter
  • Ingår i: FEBS Letters. - : Wiley-Blackwell. - 0014-5793. ; 526, s. 31-37
  • Tidskriftsartikel (refereegranskat)abstract
    • A widely accepted mechanism for selective degradation of plasma membrane proteins is via ubiquitination and/or phosphorylation events. Such a regulated degradation has previously been suggested to rely on the presence of a specific SINNDAKSS sequence within the protein. Modification of a partly conserved SINNDAKSS-like sequence in the C-terminal tail of the Pho84 phosphate transporter, in combination with C-terminal fusion of green fluorescent protein or a MYC epitope, were used to evaluate the presence of this sequence and its role in the regulated degradation. The functional Pho84 mutants in which this SINNDAKSS-like sequence was altered or truncated were subjected to degradation like that of the wild type, suggesting that degradation of the Pho84 protein is regulated by factors other than properties of this sequence.
  • Lundh, Fredrik, et al. (författare)
  • Molecular mechanisms controlling phosphate-induced downregulation of the yeast Pho84 phosphate transporter
  • Ingår i: Biochemistry. - : The American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 48:21, s. 505-4497
  • Tidskriftsartikel (refereegranskat)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.
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