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

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.

Fig. 1.

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.

Fig. 2.

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).

Fig. 3.

Concentration dependence of the mβcd-induced depletion of proteins from Arabidopsis plasma membrane. The graph shows the average log₂ value (±S.D.) of the ¹⁵N to ¹⁴N abundance ratios for proteins identified in the supernatant after pelleting of treated versus untreated plasma membranes. Only proteins identified as mβcd-responsive in reciprocal experiments have been considered (n = 37).

Fig. 4.

The effect of mβcd treatment on sterol composition of Arabidopsis plasma membrane. A, abundance of different sterols relative to the standard 3β-hydroxy-5α-cholestane. B, relative changes in sterol content of plasma membrane upon treatment with mβcd at 15 or 30 mm for 1 h. Averages ± S.D. of three biological replicates are shown. d5avenasterol, Δ⁵-avenasterol; d7avenasterol, Δ⁷-avenasterol.

Fig. 5.

A, distribution of mβcd-responsive proteins (blue) and proteins without an mβcd response (red) to different functional categories. Proteins were classified according to the MapMan classification scheme for plant proteins (34). n = 74 for sterol-dependent proteins and n = 295 for non-responsive proteins. B, subcellular localization from mass spectrometry and/or GFP localization of proteins identified in the mβcd disruption experiments. Subcellular localization was obtained from the SUBA database. RLKs, receptor-like kinases; ABC, ATP-binding cassette; cw, cell wall.

Fig. 6.

A box plot of the deviations from the mean ratio change upon mβcd treatment for each protein in the functional categories “cell wall-related,” “lipid-modifying,” and “vesicle trafficking” as well as for signaling proteins in “receptor kinases,” “calcium signaling,” and “G-protein signaling” across four independent reciprocal experiments is shown. Proteins were classified according to the MapMan classification scheme for plant proteins (34).