Predicted protein sequence of AtRGP1 (GenBank accession no. AF013627) and comparison with other RGP proteins. A, Amino acid sequence alignment of AtRGP1, PsRGP1 (U31565), VuRGP (AF005279), ZmRGP (U89897), and OsRGP using the MegAlign module of DNAStar. The black regions represent amino acid differences between AtRGP1 and the other RGPs. The overline highlights a putative N-myristoylation site. The shaded box represents the predicted UDP-Glc-binding domain U1 from cotton CelA. The open box is the site of glycosylation as determined in sweet corn. Underlined are the two pea tryptic peptides used to search the EST database. B, Alignment of UDP-Glc-binding domains from various organisms, as determined by Delmer and Amor (1995), with the predicted binding domain of RGP.

Homologs of AtRGP1 from other species. Total protein (50 μg) from Arabidopsis (lane 1), pea (lane 2), tobacco (lane 3), maize (lane 4), cyanobacteria (lane 5), yeast (lane 6), and HeLa cells (lane 7) was analyzed by immunoblot using anti-AtRGP1 antibodies. All plant-derived protein extracts were obtained from root tissue. Molecular mass is indicated in kilodaltons.

AtRGP1 RNA and protein are highest in suspension-cultured cells and roots from whole plants. A, RNA levels in different tissues. Total RNA (30 μg) from A. thaliana flowers (lane 1), leaves (lane 2), roots (lane 3), stems (lane 4), and suspension-cultured cells (lane 5) was separated in a 1% agarose and 6% formaldehyde gel, transferred onto a nylon membrane, and hybridized with a random-primed, ³²P-labeled, 1.0-kb EcoRI-BbvI fragment of the AtRGP1 cDNA. The single band obtained is of the expected size. Molecular mass is indicated in kilodaltons. B, Protein levels in different tissues. Immunoprecipitations using total protein from [³⁵S]Met-labeled protoplasts (lane 1) and unlabeled protoplasts (lane 2) were performed. The former was analyzed by SDS-PAGE and autoradiography and the latter was analyzed by immunoblot. Total protein (50 μg) from A. thaliana flowers (lane 3), leaves (lane 4), roots (lane 5), stems (lane 6), root liquid culture (lane 7), and cell-suspension cultures (lane 8) was analyzed by immunoblotting using anti-AtRGP1 antibodies. Molecular mass is indicated in kilodaltons. C, AtRGP1 RNA and protein levels during the growth cycle of suspension cells. A cell-suspension culture was started by diluting a 1-week-old culture 10 times with fresh medium. Samples representing 1/40 of the original volume were collected at 1-d intervals, and protein and RNA were extracted from each sample. Top, Total AtRGP1 protein from each sample was analyzed by immunoblot; bottom, total AtRGP1 RNA was determined as described in Figure 4A.

AtRGP1 is soluble and membrane associated. A, Total protoplast protein from suspension-cultured cell protoplasts was centrifuged at 1,000, 5,000, 10,000, 15,000, 25,000, 50,000, and 100,000g. The resultant pellets were resuspended in lysis buffer and make up fractions p1, p5, p10, p15, p25, p50, and p100, respectively. s100 denotes the supernatant after the 100,000g centrifugation and represents total soluble proteins. Equal volumes of protein were separated by SDS-PAGE, transferred to nitrocellulose, and analyzed by immunoblot. Various soluble proteins (BiP), integral membrane proteins (AtPEP12p, RD28, and AtELP), and peripheral membrane proteins (ARA4) were compared with the membrane association of AtRGP1. The fraction of total protein (T) present in each pellet is shown. B, AtRGP1 is a peripheral membrane protein. Total microsomes were prepared by centrifuging total protein at 150,000g for 1 h and washing the pellet with lysis buffer. Pellets were resuspended with various buffers for 1 h on ice and centrifuged again at 150,000g for 1 h, and the pellets were analyzed by immunoblot using anti-AtRGP1 antibodies. Microsomes were resuspended with lysis buffer (lane 1, total protein) or lysis buffer containing 0.1, 0, or 1.0% Triton X-100 (lanes 2, 3, and 4, respectively), 0.5 or 2 m urea (lanes 5 and 6, respectively), 0.1 m sodium carbonate (lane 7), 0.1, 0.5, or 1.0 m NaCl (lanes 8, 9, and 10, respectively), or 0.1 m potassium phosphate buffer, pH 7.0, alone (lane 11).

Membrane localization of AtRGP1. Equilibrium-density gradients were used to fractionate A. thaliana total membrane preparations from suspension-cultured cell protoplasts. One-twentieth (100 μL) of equal-volume fractions was separated by SDS-PAGE and transferred to nitrocellulose membranes. A, Fractionation of AtRGP1 was determined by immunoblot analysis and compared with that of the membrane marker BiP (ER). Because of the presence of AtRGP1 and BiP in the same fractions, similar gradients were analyzed in the presence of Mg⁺² and showed that BiP shifted to a denser part of the gradient, but AtRGP1 did not (data not shown). B, AtRGP1 fractionation was also compared with that of other known membrane markers, AtELP, AtPEP12p, and R28.

AtRGP1 is reversibly autoglycosylated. A, Total protein (100–250 μg) from Arabidopsis (lane 1), pea (lane 2), tobacco (lane 3), maize (lane 4), and cyanobacteria (lane 5); 5 μg of purified GST (lane 6) and GST-AtRGP1p (lane 7) and 50 μg of the Arabidopsis Suc-gradient fraction 10 (lane 8) were incubated with 0.1 μCi of UDP-[¹⁴C]Glc before analysis by SDS-PAGE and autoradiography. Molecular mass is indicated in kilodaltons. B, Displacement of bound radiolabel from GST-AtRGP1. Two micrograms of GST-AtRGP1 was incubated with UDP-[³H]Glc for 10 min. Various substrates were then added to a final concentration of 3 mm and the incubations were continued for 10 min more. Lane 1, No substrate added; lane 2, UDP; lane 3, Glc; lane 4, UDP-Glc, lane 5, UDP-Xyl; lane 6, UDP-Gal; and lane 7, UDP-Man. After the second incubation, reactions were stopped and analyzed by SDS-PAGE and autoradiography.