One-Dimensional Thin-Layer Chromatography of All Known D-3 and D-4 Isomers of Phosphoinositides

Hegewald, H

doi:10.1006/abio.1996.0443

Cited by 45 publications

(16 citation statements)

References 1 publication

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“…The radiolabeled lipid spot in Figure 7A was identified as PtdInsP as it co-migrated with PtdIns(4)P but not with PtdIns(4,5)P 2 or PtdIns(3,4,5)P 3 reference lipid-standards (data not shown). PtdIns(4)P and PtdIns(3)P migrate with the same R f value and are not resolved with this assay system [58], [59]. Lysates from cells carrying the wild-type pik1 allele had higher incorporation of 32 P label into PtdInsP when expression was derepressed (Figure 7A; WT, compare + and − thiamine).…”

Section: Resultsmentioning

confidence: 97%

Essential Role for Schizosaccharomyces pombe pik1 in Septation

et al. 2009

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Background Schizosaccharomyces pombe pik1 encodes a phosphatidylinositol 4-kinase, reported to bind Cdc4, but not Cdc4G107S.Principal FindingsGene deletion revealed that pik1 is essential. In cells with pik1 deleted, ectopic expression of a loss-of-function allele, created by fusion to a temperature-sensitive dihydrofolate reductase, allowed normal cell proliferation at 25°C. At 36°C, cells arrested with abnormally thick, misplaced or supernumerary septa, indicating a defect late in septation. In addition to being Golgi associated, ectopically expressed GFP-tagged Pik1 was observed at the medial cell plane late in cytokinesis. New alleles, created by site-directed mutagenesis, were expressed ectopically. Lipid kinase and Cdc4-binding activity assays were performed. Pik1D709A was kinase-dead, but bound Cdc4. Pik1R838A did not bind Cdc4, but was an active kinase. Genomic integration of these substitutions in S. pombe and complementation studies in Saccharomyces cerevisiae pik1-101 cells revealed that D709 is essential in both cases while R838 is dispensable. In S. pombe, ectopic expression of pik1 was dominantly lethal; while, pik1D709A,R838A was innocuous, pik1R838A was almost innocuous, and pik1D709A produced partial lethality and septation defects. The pik1 ectopic expression lethal phenotype was suppressed in cdc4G107S. Thus, D709 is essential for kinase activity and septation.ConclusionsPik1 kinase activity is required for septation. The Pik1 R838 residue is required for important protein-protein interactions, possibly with Cdc4.

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Section: Resultsmentioning

confidence: 97%

Essential Role for Schizosaccharomyces pombe pik1 in Septation

et al. 2009

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“…As a consequence, we needed to discriminate between the different PtdIns 3-, 4-, or 5-kinase and PtdInsP 4-or 5-kinase activities (Mueller-Roeber and Pical, 2002). Using an appropriate HP-TLC solvent system, we first determined the nature of PtdInsP isomers synthesized from exogenously added PtdIns in tobacco PM (Hegewald, 1996). Only PtdIn4P and PtdIns3P were synthesized, and no PtdIn5P was detected (Supplemental Fig.…”

Section: Activities Of Signaling Lipid-modifying Enzymes Are Present mentioning

confidence: 99%

Polyphosphoinositides Are Enriched in Plant Membrane Rafts and Form Microdomains in the Plasma Membrane

et al. 2010

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In this article, we analyzed the lipid composition of detergent-insoluble membranes (DIMs) purified from tobacco (Nicotiana tabacum) plasma membrane (PM), focusing on polyphosphoinositides, lipids known to be involved in various signal transduction events. Polyphosphoinositides were enriched in DIMs compared with whole PM, whereas all structural phospholipids were largely depleted from this fraction. Fatty acid composition analyses suggest that enrichment of polyphosphoinositides in DIMs is accompanied by their association with more saturated fatty acids. Using an immunogold-electron microscopy strategy, we were able to visualize domains of phosphatidylinositol 4,5-bisphosphate in the plane of the PM, with 60% of the epitope found in clusters of approximately 25 nm in diameter and 40% randomly distributed at the surface of the PM. Interestingly, the phosphatidylinositol 4,5-bisphosphate cluster formation was not significantly sensitive to sterol depletion induced by methyl-b-cyclodextrin. Finally, we measured the activities of various enzymes of polyphosphoinositide metabolism in DIMs and PM and showed that these activities are present in the DIM fraction but not enriched. The putative role of plant membrane rafts as signaling membrane domains or membrane-docking platforms is discussed.

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“…To examine whether the two enzyme forms catalyzed the phosphorylation of PtdIns in the 3-or 4-position, the reaction products were separated by TLC in the presence of borate (Walsh et al, 1991;Hegewald, 1996) and compared with the products obtained with PtdIns 3-kinase from rat liver cytosol (Fig. 7).…”

Section: Ptdins Phosphorylation Sitementioning

confidence: 99%

Phosphatidylinositol 4-Kinase Associated with Spinach Plasma Membranes. Isolation and Characterization of Two Distinct Forms

et al. 1999

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Highly purified plasma membranes from spinach (Spinacia oleracea L.) leaves contained phosphatidylinositol (PtdIns) kinase activity that was firmly associated with the membrane. The enzyme was solubilized by detergent treatment (2% [w/v] Triton X-100) and purified by heparin-Sepharose and Q-Sepharose chromatography. Two enzymically active fractions, QI and QII, both exhibiting PtdIns 4-kinase activity, were resolved and purified 100-to 300-fold over the plasma membrane. QI and QII shared similar high apparent K m values for ATP (approximately 0.45 mM) and PtdIns (approximately 0.2 mM) and were insensitive to inhibition by adenosine. While Mg 2؉ was the preferred divalent cation, Mn 2؉ could partly substitute in the reaction catalyzed by the QII enzyme but not in that catalyzed by QI. Mn 2؉ acted synergistically with suboptimal Mg 2؉ concentrations to activate not only the QII enzyme, but also to some extent QI. Both enzymes were inhibited by millimolar concentrations of Ca 2؉ and micromolar concentrations of wortmannin. The apparent molecular mass for QI was 120 kD, which was determined by SDS-PAGE and western blotting using an antibody against a peptide unique for lipid kinases and the binding of 3 H-wortmannin, and for QII 65 kD as determined by immunodetection and renaturation of PtdIns kinase activity in the 65-kD region of polyacrylamide gels.

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One-Dimensional Thin-Layer Chromatography of All Known D-3 and D-4 Isomers of Phosphoinositides

Cited by 45 publications

References 1 publication

Essential Role for Schizosaccharomyces pombe pik1 in Septation

Essential Role for Schizosaccharomyces pombe pik1 in Septation

Polyphosphoinositides Are Enriched in Plant Membrane Rafts and Form Microdomains in the Plasma Membrane

Phosphatidylinositol 4-Kinase Associated with Spinach Plasma Membranes. Isolation and Characterization of Two Distinct Forms

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