CTP-dependent lipid kinases of yeast

Szkopińska, Anna; Nowak, L; Świeżewska, Ewa; Palamarczyk, Grażyna

doi:10.1016/0003-9861(88)90242-1

Cited by 23 publications

(25 citation statements)

References 25 publications

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“…Lipid Kinase Assay and Two-phase Extraction of Membrane LipidsEnvelope membranes (200 g) were incubated in the presence of 10 Ci of [␥- 32 P]ATP (3000 Ci/mmol), 5 mM MgCl 2 , and 50 mM Mops (pH 7.6) at 25°C for 5 min in a final volume of 200 l. The lipid kinase reaction was stopped by mixing the samples with 1.5 ml of cold chloroform/methanol (1:2). After adding the chloroform/methanol to phosphorylated envelopes, 100 nmol of PA (Sigma), 100 nmol of LPA (Sigma), and 600 nmol of a phospholipid mixture containing PIP 2 , PIP, phosphatidylinositol, and phosphatidylserine (Sigma) were added as carriers.…”

Section: Methodsmentioning

confidence: 99%

“…They were originally found after 32 P labeling of envelope membranes using a mixture of carrier-free [␥- 32 Fig. 1B, the lipid phosphorylation took place in envelope membranes exclusively.…”

Section: Lipid Phosphorylation In Chloroplast Envelopementioning

confidence: 98%

“…Indeed, under both conditions, PA, LPA, X, and Y were labeled, therefore indicating that both chloroplastic and exogenous sources of ATP can generate phosphorylated lipids. In contrast, labeled phosphatidylinositol derivatives comigrating with LPIP, PIP, and PIP 2 used [␥- 32 (26). To determine whether CDP and UDP were labeled in intact chloroplasts in the absence of exogenous NDPs, the experiments were repeated using intact chloroplasts incubated in the presence of Fig.…”

Section: Lipid Phosphorylation In Chloroplast Envelopementioning

confidence: 99%

“…In lysed chloroplasts (data not shown), the synthesis of [␥-32 P]CTP was strongly reduced compared with intact organelles, suggesting that the integrity of the plastid is necessary for this nucleotide phosphotransferase activity. These experiments and the knowledge that CTP is also a specific substrate for farnesol and dolichol phosphorylation (32,33) encouraged us to investigate whether CTP also plays a role in the envelope lipid phosphorylation. The incubation of envelope membranes in the presence of purified NDPK-II (34) (as the phosphotransferase), [␥- 32 P]ATP, and CDP was performed (Fig.…”

Section: Lipid Phosphorylation In Chloroplast Envelopementioning

confidence: 99%

“…These experiments and the knowledge that CTP is also a specific substrate for farnesol and dolichol phosphorylation (32,33) encouraged us to investigate whether CTP also plays a role in the envelope lipid phosphorylation. The incubation of envelope membranes in the presence of purified NDPK-II (34) (as the phosphotransferase), [␥- 32 P]ATP, and CDP was performed (Fig. 2B) 32 P]ATP (lane 2) were used as labeled nucleotides.…”

Section: Lipid Phosphorylation In Chloroplast Envelopementioning

confidence: 99%

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Lipid Phosphorylation in Chloroplast Envelopes

Müller¹,

Meylan-Bettex²,

Eckstein³

et al. 2000

Journal of Biological Chemistry

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Lipid phosphorylation takes place within the chloroplast envelope. In addition to phosphatidic acid, phosphatidylinositol phosphate, and their corresponding lyso-derivatives, we found that two novel lipids underwent phosphorylation in envelopes, particularly in the presence of carrier-free [ The lipid kinase activity involved in this reaction was specifically inhibited in the presence of cytosine 5-O-(thiotriphosphate) (CTP␥S) and sensitive to CTP chase, thereby showing that both lipids are phosphorylated by an envelope CTP-dependent lipid kinase. The lipids were identified as phosphorylated galactolipids by using an acid hydrolysis procedure that generated galactose 6-phosphate. CTP␥S did not affect the import of the small ribulose-bisphosphate carboxylase/oxygenase subunit into chloroplasts, the possible physiological role of this novel CTP-dependent galactolipid kinase activity in the chloroplast envelope is discussed.One of the main functions assigned to the chloroplast is its ability to assimilate CO 2 under illumination. This key activity must be separated from the rest of the cell by a selective membrane barrier, namely the envelope, which is one of the three main plastid compartments in addition to thylakoids and the stroma. The envelope is constituted of two membranes, the inner and the outer envelopes, each having its own specific characteristic and property (1).Lipid phosphorylation occurs in chloroplast envelope membranes (2). The first identified lipids incorporating phosphate from [␥-32 P]ATP in isolated envelope vesicles were lysophosphatidic acid (LPA), 1 phosphatidic acid (PA), phosphatidylinositol phosphate (PIP), and lysophosphatidylinositol phosphate (LPIP). At the present time, the functions of these phospholipids are not known in chloroplasts, but: (i) they might produce substrates for lipid biosynthesis pathways; for instance, PIP can serve as substrate for a phosphatidylinositol-4,5-phosphate kinase and eventually can be hydrolyzed in second messengers like inositol 1,4,5-trisphosphate and DAG (3); (ii) they could directly interact with intracellular proteins to affect their location and/or activity; (iii) they might also change the local topology of other lipids, thereby modifying electrostatic interactions of membrane components. Indeed, in mammalian systems, LPA and PA, as minor cell lipids, are likely to act as potent activators of plasma membrane tyrosine kinase via G protein activation and intracellular protein kinases (4, 5). PA is the product of sn-diacylglycerol phosphorylation catalyzed by a diacylglycerol kinase (6). In plant signaling pathways, PA is also derived from PC via phospholipase D (7) and from diacylglycerol pyrophosphate (8). The production of LPA usually occurs after a first rapid accumulation of DAG, followed by phosphorylation, and activation of a PA-specific phospholipase A 2 within the plasma membrane (9). In plants, a diacylglycerol kinase from Arabidopsis thaliana (10) and LPA, a product of inducible PLA 2 (11), have been identified, but their specific role a...

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Section: Methodsmentioning

confidence: 99%

Section: Lipid Phosphorylation In Chloroplast Envelopementioning

confidence: 98%

Section: Lipid Phosphorylation In Chloroplast Envelopementioning

confidence: 99%

Section: Lipid Phosphorylation In Chloroplast Envelopementioning

confidence: 99%

Section: Lipid Phosphorylation In Chloroplast Envelopementioning

confidence: 99%

See 3 more Smart Citations

Lipid Phosphorylation in Chloroplast Envelopes

Müller¹,

Meylan-Bettex²,

Eckstein³

et al. 2000

Journal of Biological Chemistry

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A Novel Design Method of Ratiometric Fluorescent Probes Based on Fluorescence Resonance Energy Transfer Switching by Spectral Overlap Integral

Takakusa

Kikuchi

Urano

et al. 2003

Chemistry A European J

122

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A ratiometric measurement, namely, simultaneous recording of the fluorescence intensities at two wavelengths and calculation of their ratio, allows greater precision than measurements at a single wavelength, and is suitable for cellular imaging studies. Here we describe a novel method of designing probes for ratiometric measurement of hydrolytic enzyme activity based on switching of fluorescence resonance energy transfer (FRET). This method employs fluorescent probes with a 3'-O,6'-O-protected fluorescein acceptor linked to a coumarin donor through a linker moiety. As there is no spectral overlap integral between the coumarin emission and fluorescein absorption, the fluorescein moiety cannot accept the excitation energy of the donor moiety and the donor fluorescence can be observed. After cleavage of the protective groups by hydrolytic enzymes, the fluorescein moiety shows a strong absorption in the coumarin emission region, and then acceptor fluorescence due to FRET is observed. Based on this mechanism, we have developed novel ratiometric fluorescent probes (1-3) for protein tyrosine phosphatase (PTP) activity. They exhibit a large shift in their emission wavelength after reaction with PTPs. The fluorescence quenching problem that usually occurs with FRET probes is overcome by using the coumarin-cyclohexane-fluorescein FRET cassette moiety, in which close contact of the two dyes is hindered. After study of their chemical and kinetic properties, we have concluded that compounds 1 and 2 bearing a rigid cyclohexane linker are practically useful for the ratiometric measurement of PTPs activity. The design concept described in this paper, using FRET switching by spectral overlap integral and a rigid link that prevents close contact of the two dyes, should also be applicable to other hydrolytic enzymes by introducing other appropriate enzyme-cleavable groups into the fluorescein acceptor.

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Dissecting the role of dolichol in cell wall assembly in the yeast mutants impaired in early glycosylation reactions

Orłowski

Machula

Janik

et al. 2007

Yeast

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Evidence is presented that temperature-sensitive Saccharomyces cerevisiae mutants, impaired in dolichol kinase (Sec59p) or dolichyl phosphate mannose synthase (Dpm1p) activity have an aberrant cell wall composition and ultrastructure. The mutants were oversensitive to Calcofluor white, an agent interacting with the cell wall chitin. In accordance with this, chemical analysis of the cell wall alkali-insoluble fraction indicated an increased amount of chitin and changes in the quantity of β1,6-and β1,3-glucan in sec59-1 and dpm1-6 mutants. In order to unravel the link between the formation of dolichyl phosphate and dolichyl phosphate mannose and the cell wall assembly, we screened a yeast genomic library for a multicopy suppressors of the thermosensitive phenotype. The RER2 and SRT1 genes, encoding cis-prenyltransferases, were isolated. In addition, the ROT1 gene, encoding protein involved in β1,6-glucan synthesis (Machi et al., 2004) and protein folding (Takeuchi et al., 2006) acted as a multicopy suppressor of the temperature-sensitive phenotype of the sec59-1 mutant. The cell wall of the mutants and of mutants bearing the multicopy suppressors was analysed for carbohydrate and mannoprotein content. We also examined the glycosylation status of the plasma membrane protein Gas1p, a β1,3-glucan elongase, and the degree of phosphorylation of the Mpk1/Slt2 protein, involved in the cell wall integrity pathway.

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CTP-dependent lipid kinases of yeast

Cited by 23 publications

References 25 publications

Lipid Phosphorylation in Chloroplast Envelopes

Lipid Phosphorylation in Chloroplast Envelopes

A Novel Design Method of Ratiometric Fluorescent Probes Based on Fluorescence Resonance Energy Transfer Switching by Spectral Overlap Integral

Dissecting the role of dolichol in cell wall assembly in the yeast mutants impaired in early glycosylation reactions

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