Figure 4.

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).