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Definition of Arabidopsis sterol-rich membrane microdomains by differential treatment with methyl-beta-cyclodextrin and quantitative proteomics.

Kierszniowska S, Seiwert B, Schulze WX - Mol. Cell Proteomics (2008)

Bottom Line: Among the sterol-dependent proteins we found an over-representation of glycosylphosphatidylinositol-anchored proteins.Predominantly proteins with signaling functions, such as receptor kinases, G-proteins, and calcium signaling proteins, were identified as variable members in plant lipid rafts, whereas cell wall-related proteins and specific proteins with unknown functions make up a core set of sterol-dependent plant plasma membrane proteins.This allows the plant to maintain a balance between static anchoring of cell shape forming elements and variable adjustment to changing external conditions.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut für Molekulare Pflanzenphysiologie, Am Mühlenberg 1, 14476 Golm, Germany.

ABSTRACT
Plasma membranes are dynamic compartments with key functions in solute transport, cell shape, and communication between cells and the environment. In mammalian cells and yeast, the plasma membrane has been shown to be compartmented into so-called lipid rafts, which are defined by their resistance to treatment with non-ionic detergents. In plants, the existence of lipid rafts has been postulated, but the precise composition of this membrane compartment is still under debate. Here we were able to experimentally clearly distinguish (i) true sterol-dependent "raft proteins" and (ii) sterol-independent "non-raft" proteins and co-purifying "contaminants" in plant detergent-resistant membranes. We used quantitative proteomics techniques involving (15)N metabolic labeling and specific disruption of sterol-rich membrane domains by methyl-beta-cyclodextrin. Among the sterol-dependent proteins we found an over-representation of glycosylphosphatidylinositol-anchored proteins. A large fraction of these proteins has functions in cell wall anchoring. We were able to distinguish constant and variable components of plant sterol-rich membrane microdomains based on their responsiveness to the drug methyl-beta-cyclodextrin. Predominantly proteins with signaling functions, such as receptor kinases, G-proteins, and calcium signaling proteins, were identified as variable members in plant lipid rafts, whereas cell wall-related proteins and specific proteins with unknown functions make up a core set of sterol-dependent plant plasma membrane proteins. This allows the plant to maintain a balance between static anchoring of cell shape forming elements and variable adjustment to changing external conditions.

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Work flow of the reciprocal labeling experiments. Two experiments were carried out in parallel. In one case, the 14N cells were subjected to the mβcd treatment, and 15N cells were used as control. In the second case, the 15N cells were used for mβcd treatment, whereas the 14N cells were used as untreated control. In addition, 1:1 mixtures of untreated 14N and 15N cells were used to define inherent differences between the cell cultures and the technical variation. The complete reciprocal experimental design was repeated four times independently. PM, plasma membrane.
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f1: Work flow of the reciprocal labeling experiments. Two experiments were carried out in parallel. In one case, the 14N cells were subjected to the mβcd treatment, and 15N cells were used as control. In the second case, the 15N cells were used for mβcd treatment, whereas the 14N cells were used as untreated control. In addition, 1:1 mixtures of untreated 14N and 15N cells were used to define inherent differences between the cell cultures and the technical variation. The complete reciprocal experimental design was repeated four times independently. PM, plasma membrane.

Mentions: For comparative proteomics analysis, plasma membranes of 15N-labeled cell cultures were treated with mβcd, whereas plasma membranes of control cells were left untreated. In a paired reciprocal experiment using the same starting material, 14N cells were treated with mβcd, whereas 15N cells were left untreated. Labeled and unlabeled plasma membranes were treated with Triton X-100 in one combined sample prior to DRM preparation over a sucrose gradient. The work flow of the experimental setup is shown in Fig. 1. The paired reciprocal experiments (Fig. 1) were independently repeated four times. Protein ratios of each replicate experimental set were z-score-standardized to allow comparison between experiments. In result tables and figures, the averages and S.D. of z-transformed protein ratios from all biological replicate experiments are presented. In general, 60% of all identified proteins were found in at least two independent reciprocal experimental setups, and 25% of all identified proteins were found in all four replicate experiments (supplemental Fig. 2).


Definition of Arabidopsis sterol-rich membrane microdomains by differential treatment with methyl-beta-cyclodextrin and quantitative proteomics.

Kierszniowska S, Seiwert B, Schulze WX - Mol. Cell Proteomics (2008)

Work flow of the reciprocal labeling experiments. Two experiments were carried out in parallel. In one case, the 14N cells were subjected to the mβcd treatment, and 15N cells were used as control. In the second case, the 15N cells were used for mβcd treatment, whereas the 14N cells were used as untreated control. In addition, 1:1 mixtures of untreated 14N and 15N cells were used to define inherent differences between the cell cultures and the technical variation. The complete reciprocal experimental design was repeated four times independently. PM, plasma membrane.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2667346&req=5

f1: Work flow of the reciprocal labeling experiments. Two experiments were carried out in parallel. In one case, the 14N cells were subjected to the mβcd treatment, and 15N cells were used as control. In the second case, the 15N cells were used for mβcd treatment, whereas the 14N cells were used as untreated control. In addition, 1:1 mixtures of untreated 14N and 15N cells were used to define inherent differences between the cell cultures and the technical variation. The complete reciprocal experimental design was repeated four times independently. PM, plasma membrane.
Mentions: For comparative proteomics analysis, plasma membranes of 15N-labeled cell cultures were treated with mβcd, whereas plasma membranes of control cells were left untreated. In a paired reciprocal experiment using the same starting material, 14N cells were treated with mβcd, whereas 15N cells were left untreated. Labeled and unlabeled plasma membranes were treated with Triton X-100 in one combined sample prior to DRM preparation over a sucrose gradient. The work flow of the experimental setup is shown in Fig. 1. The paired reciprocal experiments (Fig. 1) were independently repeated four times. Protein ratios of each replicate experimental set were z-score-standardized to allow comparison between experiments. In result tables and figures, the averages and S.D. of z-transformed protein ratios from all biological replicate experiments are presented. In general, 60% of all identified proteins were found in at least two independent reciprocal experimental setups, and 25% of all identified proteins were found in all four replicate experiments (supplemental Fig. 2).

Bottom Line: Among the sterol-dependent proteins we found an over-representation of glycosylphosphatidylinositol-anchored proteins.Predominantly proteins with signaling functions, such as receptor kinases, G-proteins, and calcium signaling proteins, were identified as variable members in plant lipid rafts, whereas cell wall-related proteins and specific proteins with unknown functions make up a core set of sterol-dependent plant plasma membrane proteins.This allows the plant to maintain a balance between static anchoring of cell shape forming elements and variable adjustment to changing external conditions.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut für Molekulare Pflanzenphysiologie, Am Mühlenberg 1, 14476 Golm, Germany.

ABSTRACT
Plasma membranes are dynamic compartments with key functions in solute transport, cell shape, and communication between cells and the environment. In mammalian cells and yeast, the plasma membrane has been shown to be compartmented into so-called lipid rafts, which are defined by their resistance to treatment with non-ionic detergents. In plants, the existence of lipid rafts has been postulated, but the precise composition of this membrane compartment is still under debate. Here we were able to experimentally clearly distinguish (i) true sterol-dependent "raft proteins" and (ii) sterol-independent "non-raft" proteins and co-purifying "contaminants" in plant detergent-resistant membranes. We used quantitative proteomics techniques involving (15)N metabolic labeling and specific disruption of sterol-rich membrane domains by methyl-beta-cyclodextrin. Among the sterol-dependent proteins we found an over-representation of glycosylphosphatidylinositol-anchored proteins. A large fraction of these proteins has functions in cell wall anchoring. We were able to distinguish constant and variable components of plant sterol-rich membrane microdomains based on their responsiveness to the drug methyl-beta-cyclodextrin. Predominantly proteins with signaling functions, such as receptor kinases, G-proteins, and calcium signaling proteins, were identified as variable members in plant lipid rafts, whereas cell wall-related proteins and specific proteins with unknown functions make up a core set of sterol-dependent plant plasma membrane proteins. This allows the plant to maintain a balance between static anchoring of cell shape forming elements and variable adjustment to changing external conditions.

Show MeSH