Abstract
Arabinogalactan proteins constitute a class of plant cell surface proteoglycans with widespread occurrence and suggested functions in various aspects of plant growth and development, including cell proliferation, expansion, marking, and death. Previous investigations of subcellular fractions from suspension-cultured cells of "Paul's Scarlet" rose (Rosa sp.) have revealed extensive structural similarity between some soluble arabinogalactan proteins from the cell wall space and some plasma membrane-associated arabinogalactan proteins, thus inspiring the present investigation of the mechanism through which these inherently water-soluble molecules are held on the plasma membrane. Several lines of evidence gained through a combination of methods including reversed-phase chromatography, treatment with phosphatidylinositol-specific phospholipase C, and chemical structural analysis now show that some rose arabinogalactan proteins carry a ceramide class glycosylphosphatidylinositol lipid anchor. The predominant form of the ceramide is composed of tetracosanoic acid and 4-hydroxysphinganine. Plasma membrane vesicles readily shed arabinogalactan proteins by an inherent mechanism that appears to involve a phospholipase. This finding has significance toward understanding the biosynthesis, localization, and function of arabinogalactan proteins and toward stimulating other studies that may expand the currently very short list of higher plant proteins found to carry such membrane lipid anchors.
Highlights
Through biochemical purification and chemical structural analysis, PM-Arabinogalactan proteins (AGPs) bound to the plasma membrane, CW-AGPs bound to the cell wall, and soluble CM-AGPs of the cell wall space/culture medium have been characterized in this model system [17,18,19]
The hypothesis that PM-AGPs contain a GPI lipid anchor was tested by quantitating the release of AGPs from plasma membrane vesicles upon treatment with PI-PLC
These results showed that treatment with PI-PLC reduced the hydrophobicity of PMAGP-III, consistent with the hypothesis that this AGP contains a GPI lipid anchor
Summary
Purification of AGPs—Total PM-AGPs were purified from suspension-cultured cells of Paul’s Scarlet rose (Rosa sp.) as described [19]. Analysis of AGPs by RPC—In typical experiments, samples of CM- or PM-AGPs (up to 1.7 mg of carbohydrate dissolved in water up to 300 l) were analyzed on a 3-ml Resource-RPC column (Amersham Pharmacia Biotech) using equipment and elution with gradients of acetonitrile and water containing 0.1% (v/v) trifluoroacetic acid as described [19]. In experiments involving enzyme treatment of AGPs prior to RPC separation, CM- or PM-AGPs (0.09 –1.7 mg of total carbohydrate) were incubated with 3.5– 4 units of PI-PLC in 200 –300 l of 50 mM Tris-HCl (pH 7.5) for 3 h at 37 °C. For analysis of fatty acid methyl esters, water was added to methanolysates to achieve a 4:1 (v/v) methanol-water ratio, and the mixture was extracted three times, each with two volumes of hexane. Analyses of aminoacyl compositions were performed at the Macromolecular Structure Facility at the Michigan State University
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