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Subcellular Localization of Carotenoid Biosynthesis in Synechocystis sp. PCC 6803.

Zhang L, Selão TT, Selstam E, Norling B - PLoS ONE (2015)

Bottom Line: Carotenoid analysis of different membrane subfractions in Synechocystis sp.PCC6803 shows that "light" plasma membranes have a high carotenoid/protein ratio, when compared to "heavier" plasma membranes or thylakoids.Both enzymes are locally more abundant in plasma membranes than in thylakoids, implying that the plasma membrane has higher synthesis rates of β-carotene precursor molecules and echinenone.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, Nanyang Technological University, Singapore.

ABSTRACT
The biosynthesis pathway of carotenoids in cyanobacteria is partly described. However, the subcellular localization of individual steps is so far unknown. Carotenoid analysis of different membrane subfractions in Synechocystis sp. PCC6803 shows that "light" plasma membranes have a high carotenoid/protein ratio, when compared to "heavier" plasma membranes or thylakoids. The localization of CrtQ and CrtO, two well-defined carotenoid synthesis pathway enzymes in Synechocystis, was studied by epitope tagging and western blots. Both enzymes are locally more abundant in plasma membranes than in thylakoids, implying that the plasma membrane has higher synthesis rates of β-carotene precursor molecules and echinenone.

No MeSH data available.


Membrane binding analyses of CrtQ-FLAG and CrtO-FLAG.Western blots of soluble fractions vs. total membranes (5 μg each) isolated from both strains and the effect of different washes on CrtO and CrtQ membrane association. Total membranes were washed with EB8, EB12 or EB8+U and soluble (“Sol.”) and pellet (“Pel.”) fractions were generated by ultracentrifugation. Each lane was loaded with equal volumes of (resuspended) pellet or supernatant fractions. Three independent cultures were tested in this manner. (A). Western blots analysis of anti-FLAG resin eluates from 2% DDM-solubilized total membranes of WT, CrtO-FLAG and CrtQ-FLAG cells, separated on 4–18% Clear Native–PAGE (B). Colour scan of CN-PAGE gel for WT total membrane showing the relative migration of several known complexes.
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pone.0130904.g004: Membrane binding analyses of CrtQ-FLAG and CrtO-FLAG.Western blots of soluble fractions vs. total membranes (5 μg each) isolated from both strains and the effect of different washes on CrtO and CrtQ membrane association. Total membranes were washed with EB8, EB12 or EB8+U and soluble (“Sol.”) and pellet (“Pel.”) fractions were generated by ultracentrifugation. Each lane was loaded with equal volumes of (resuspended) pellet or supernatant fractions. Three independent cultures were tested in this manner. (A). Western blots analysis of anti-FLAG resin eluates from 2% DDM-solubilized total membranes of WT, CrtO-FLAG and CrtQ-FLAG cells, separated on 4–18% Clear Native–PAGE (B). Colour scan of CN-PAGE gel for WT total membrane showing the relative migration of several known complexes.

Mentions: Both CrtQ and CrtO are predicted to be soluble proteins, with some hydrophobic regions in the N-terminal, by online softwares TOPCONS (www.topcons.net) and TMHMM (http://www.cbs.dtu.dk/services/TMHMM/). These proteins are involved in the synthesis of carotenoid molecules and it is expected, from the hydrophobic nature of their products, that they should strongly associate to the membrane. Total membranes were washed with both high pH and high concentrations of urea, so as to remove membrane associated proteins, and anti-FLAG western blots of the resulting fractions showed that, though also present in the soluble fraction, neither membrane-associated CrtO nor CrtQ could be completely removed by either high pH or urea treatment, in opposition to the luminal PSII subunit PsbO (Fig 4A).


Subcellular Localization of Carotenoid Biosynthesis in Synechocystis sp. PCC 6803.

Zhang L, Selão TT, Selstam E, Norling B - PLoS ONE (2015)

Membrane binding analyses of CrtQ-FLAG and CrtO-FLAG.Western blots of soluble fractions vs. total membranes (5 μg each) isolated from both strains and the effect of different washes on CrtO and CrtQ membrane association. Total membranes were washed with EB8, EB12 or EB8+U and soluble (“Sol.”) and pellet (“Pel.”) fractions were generated by ultracentrifugation. Each lane was loaded with equal volumes of (resuspended) pellet or supernatant fractions. Three independent cultures were tested in this manner. (A). Western blots analysis of anti-FLAG resin eluates from 2% DDM-solubilized total membranes of WT, CrtO-FLAG and CrtQ-FLAG cells, separated on 4–18% Clear Native–PAGE (B). Colour scan of CN-PAGE gel for WT total membrane showing the relative migration of several known complexes.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4470828&req=5

pone.0130904.g004: Membrane binding analyses of CrtQ-FLAG and CrtO-FLAG.Western blots of soluble fractions vs. total membranes (5 μg each) isolated from both strains and the effect of different washes on CrtO and CrtQ membrane association. Total membranes were washed with EB8, EB12 or EB8+U and soluble (“Sol.”) and pellet (“Pel.”) fractions were generated by ultracentrifugation. Each lane was loaded with equal volumes of (resuspended) pellet or supernatant fractions. Three independent cultures were tested in this manner. (A). Western blots analysis of anti-FLAG resin eluates from 2% DDM-solubilized total membranes of WT, CrtO-FLAG and CrtQ-FLAG cells, separated on 4–18% Clear Native–PAGE (B). Colour scan of CN-PAGE gel for WT total membrane showing the relative migration of several known complexes.
Mentions: Both CrtQ and CrtO are predicted to be soluble proteins, with some hydrophobic regions in the N-terminal, by online softwares TOPCONS (www.topcons.net) and TMHMM (http://www.cbs.dtu.dk/services/TMHMM/). These proteins are involved in the synthesis of carotenoid molecules and it is expected, from the hydrophobic nature of their products, that they should strongly associate to the membrane. Total membranes were washed with both high pH and high concentrations of urea, so as to remove membrane associated proteins, and anti-FLAG western blots of the resulting fractions showed that, though also present in the soluble fraction, neither membrane-associated CrtO nor CrtQ could be completely removed by either high pH or urea treatment, in opposition to the luminal PSII subunit PsbO (Fig 4A).

Bottom Line: Carotenoid analysis of different membrane subfractions in Synechocystis sp.PCC6803 shows that "light" plasma membranes have a high carotenoid/protein ratio, when compared to "heavier" plasma membranes or thylakoids.Both enzymes are locally more abundant in plasma membranes than in thylakoids, implying that the plasma membrane has higher synthesis rates of β-carotene precursor molecules and echinenone.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, Nanyang Technological University, Singapore.

ABSTRACT
The biosynthesis pathway of carotenoids in cyanobacteria is partly described. However, the subcellular localization of individual steps is so far unknown. Carotenoid analysis of different membrane subfractions in Synechocystis sp. PCC6803 shows that "light" plasma membranes have a high carotenoid/protein ratio, when compared to "heavier" plasma membranes or thylakoids. The localization of CrtQ and CrtO, two well-defined carotenoid synthesis pathway enzymes in Synechocystis, was studied by epitope tagging and western blots. Both enzymes are locally more abundant in plasma membranes than in thylakoids, implying that the plasma membrane has higher synthesis rates of β-carotene precursor molecules and echinenone.

No MeSH data available.