Heterogeneity of Arabinogalactan-Proteins on the Plasma Membrane of Rose Cells

Abstract

Arabinogalactan-proteins (AGPs) have been purified from the plasma membrane of suspension-cultured Paul's Scarlet rose (Rosa sp.) cells. The two most abundant and homogeneous plasma membrane ACP fractions were named plasma membrane ACPl (PM-ACP1) and plasma membrane AGP2 (PM-ACP2) and had apparent molecular masses of 140 and 21 7 kD, respectively. Both PM-ACP1 and PM-ACP2 had p-(l-3)-, p-(1,6)-, and p-(1,3,6)-galactopyranosyl residues, predominantly terminal a-arabinofuranosyl residues, and (1,4)-and terminal glucuronopyranosyl residues. The protein moieties of PM-ACP1 and PM-ACP2 were both rich in hydroxyprolhe, alanine, and serine, but differed in the abundance of hydroxyproline, which was 1.6 times higher in PM-ACP2 than in PM-ACP1. Another difference was the overall protein content, which was 3.7% (w/w) in PM-ACP1 and 15% in PM-ACP2. As judged by their behavior on reverse-phase chromatography, PMACPl and PM-ACP2 were not more hydrophobic than ACPs from the cell wall or culture medium. In contrast, a minor plasma membrane ACP fraction eluted later on reverse-phase chromatography and was more negatively charged at pH 5 than either PM-ACP1 or PM-ACP2. The more negatively charged fraction contained molecules with a glycosyl composition characteristic of ACPs and included at least two different macromolecules. The results of this investigation indicate that Rosa plasma membrane contains at least four distinct ACPs or ACP-like molecules. These molecules differed from each other in size, charge, hydrophobicity, amino-acyl composition, and/or protein content.AGPs are a class of proteoglycans that are rich in galactosyl and arabinosyl residues Fincher et al., 1983; Bacic et al., 1988), and they appear to be present in all plant cells (Jermyn and Yeow, 1975;

Arabinogalactan-proteins: structure, expression and function

“…Thus, the model that emerges is that AGPs containing a GPI anchor are associated with the plasma membrane and are cleaved so as to release the AGP to and beyond the cell wall. Immunolocalizations as well as biochemical isolations of identical, or nearly identical AGPs (family members), in multiple surface locations (e. g. cell wall and culture media; plasma membrane, cell wall and culture media) provide evidence for multiple surface locations for a given AGP and are consistent with the above processing model for GPI-anchored AGPs [5, 10,77,78]. Nonetheless, the possibility of exclusively targeting certain AGPs to specific cell surface locations cannot be excluded, and the contribution of particular sequence determinants in the carbohydrate or protein moieties to such a process remains to be investigated.…”

Presence of a Glycosylphosphatidylinositol Lipid Anchor on Rose Arabinogalactan Proteins

“…In brief, plasma membrane vesicles were purified from the microsomal fraction by aqueous two-phase partitioning, optimized for rose (17). The PM-AGPs were fully extracted from the plasma membrane vesicles with 1% (w/w) Triton X-100 (19), and then (␤-D-Glc) 3 Yariv phenylglycoside was added to the solubilized fraction to precipitate AGPs. Named chemically as 1,3,5-tri-(p-␤-glucosyloxyphenylazo)-2,4,6-trihydroxybenzene, (␤-D-Glc) 3 selectively binds AGPs.…”

“…as described (19). In brief, plasma membrane vesicles were purified from the microsomal fraction by aqueous two-phase partitioning, optimized for rose (17).…”

“…The model system for this work has been suspension-cultured cells of "Paul's Scarlet" rose (Rosa sp.). Through biochemical purification and chemical structural analysis, PM-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). Two major AGPs, plus other minor forms, were found at each of these three sites.…”

“…As none of the PM-AGPs had aminoacyl compositions that were more hydrophobic than those of CM-AGPs (1), a membrane anchor consisting of a hydrophobic, ␣-helical polypeptide domain seemed unlikely, despite the fact that such a domain has been predicted by several cDNAs encoding core polypeptides of AGPs (3,20). Instead, the direction of this investigation was charted on the basis of our observation that the NMR spectrum of a PM-AGP included a signal of appropriate frequency and amplitude that it could have arisen from approximately two hydrocarbon chains per AGP molecule, such as might occur in a GPI or other lipid anchor (19). We now report several additional lines of evidence showing that a GPI lipid anchor does occur on some AGPs.…”