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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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A, response of proteins to mβcd treatment. Log2 values of 15N to 14N ratios from one experiment were plotted against log2 values of 15N to 14N ratios from the reciprocal experiment. Blue and red symbols indicate those sterol-dependent and sterol-independent proteins that show significant reciprocal response. Yellow symbols indicate those proteins that do not respond to the mβcd treatment (31). B, distribution of ratios from two control experiments (yellow), mβcd-responsive proteins (blue), and other proteins (red). The mβcd-responsive proteins fall into two classes: a core set of very strongly responsive (dark blue) and some less responsive ones (light blue).
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f2: A, response of proteins to mβcd treatment. Log2 values of 15N to 14N ratios from one experiment were plotted against log2 values of 15N to 14N ratios from the reciprocal experiment. Blue and red symbols indicate those sterol-dependent and sterol-independent proteins that show significant reciprocal response. Yellow symbols indicate those proteins that do not respond to the mβcd treatment (31). B, distribution of ratios from two control experiments (yellow), mβcd-responsive proteins (blue), and other proteins (red). The mβcd-responsive proteins fall into two classes: a core set of very strongly responsive (dark blue) and some less responsive ones (light blue).

Mentions: In summary, the data analysis work flow is based on first determining the variation between cultures based on 15N/14N ratios in independent 1:1 mixtures before mβcd treatment is applied. The ratios in two control experiments showed normal distribution (see Fig. 2B) and were used to define ratio-dependent standard deviations (31). In a second step, the distances to the diagonal in a graphic display of ratios in reciprocal experiments (Fig. 2A) was calculated. Then for each data point the ratio between the distance and the S.D. was calculated, and the p value was determined by a two-tailed t distribution. Subsequently a multiple testing correction was applied to the whole data set using the false discovery rate method introduced by Benjamini and Hochberg (32). Reported proteins correspond to a cutoff false discovery rate of 5%.


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)

A, response of proteins to mβcd treatment. Log2 values of 15N to 14N ratios from one experiment were plotted against log2 values of 15N to 14N ratios from the reciprocal experiment. Blue and red symbols indicate those sterol-dependent and sterol-independent proteins that show significant reciprocal response. Yellow symbols indicate those proteins that do not respond to the mβcd treatment (31). B, distribution of ratios from two control experiments (yellow), mβcd-responsive proteins (blue), and other proteins (red). The mβcd-responsive proteins fall into two classes: a core set of very strongly responsive (dark blue) and some less responsive ones (light blue).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: A, response of proteins to mβcd treatment. Log2 values of 15N to 14N ratios from one experiment were plotted against log2 values of 15N to 14N ratios from the reciprocal experiment. Blue and red symbols indicate those sterol-dependent and sterol-independent proteins that show significant reciprocal response. Yellow symbols indicate those proteins that do not respond to the mβcd treatment (31). B, distribution of ratios from two control experiments (yellow), mβcd-responsive proteins (blue), and other proteins (red). The mβcd-responsive proteins fall into two classes: a core set of very strongly responsive (dark blue) and some less responsive ones (light blue).
Mentions: In summary, the data analysis work flow is based on first determining the variation between cultures based on 15N/14N ratios in independent 1:1 mixtures before mβcd treatment is applied. The ratios in two control experiments showed normal distribution (see Fig. 2B) and were used to define ratio-dependent standard deviations (31). In a second step, the distances to the diagonal in a graphic display of ratios in reciprocal experiments (Fig. 2A) was calculated. Then for each data point the ratio between the distance and the S.D. was calculated, and the p value was determined by a two-tailed t distribution. Subsequently a multiple testing correction was applied to the whole data set using the false discovery rate method introduced by Benjamini and Hochberg (32). Reported proteins correspond to a cutoff false discovery rate of 5%.

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