FIG. 4.
Analysis of Cet1-Ceg1 interaction by glycerol gradient sedimentation. Aliquots (0.2 ml) of protein samples were applied to 4.8-ml 15 to 30% glycerol gradients containing 50 mM Tris-HCl (pH 8.0), 0.1 M NaCl, 2 mM DTT, and 0.05% Triton X-100. The gradients were centrifuged at 50,000 rpm for 13 h at 4°C in a Beckman SW50 rotor. Fractions (∼0.21 ml) were collected from the bottom of the tube (fraction 1). Aliquots (25 μl) of alternate fractions were analyzed by SDS-PAGE along with an aliquot of the material that had been applied to the gradient (lane L). The gels were fixed and stained with Coomassie blue dye. The identities of the polypeptides are indicated. (A) Sedimentation of Cet1. A 10-μg sample of the phosphocellulose enzyme fraction was applied to the glycerol gradient. Gradient fractions were assayed for RNA triphosphatase (•). The RNA triphosphatase reaction mixtures contained 20 pmol of γ-32P-labeled poly(A) and 1 μl of a 1/50 dilution of the indicated gradient fractions. Incubation was for 15 min at 30°C. (B) Sedimentation of Ceg1. A 20-μg sample of the heparin agarose Ceg1 fraction was applied to the gradient. Gradient fractions were assayed for enzyme-GMP complex formation (○). The guanylyltransferase reaction mixtures contained 0.17 μM [α-32P]GTP and 1 μl of the indicated fractions. The reaction products were analyzed by SDS-PAGE, and the signal intensity of the radiolabeled Ceg1 polypeptide (PSL, photostimulatable luminescence) was measured by scanning the dried gel with a PhosphorImager. (C) Sedimentation of a mixture of Cet1 (10 μg) and Ceg1 (20 μg). The two proteins were mixed in buffer C containing 0.1 M NaCl and then incubated on ice for 30 min before being applied to the glycerol gradient. Gradient fractions were assayed for RNA triphosphatase (•) and enzyme-GMP complex formation (○) as specified for panels A and B. The peaks of the marker proteins catalase, bovine serum albumin (BSA), and cytochrome c (cyt C), which were centrifuged in a parallel gradient, are indicated.