Figure 3.

Dynamics of TPK1-GFP assembly. Protoplasts prepared from transgenic Arabidopsis leaves were pulse labeled with a mixture of ³⁵S-Met/Cys either for 30 min (A) or for 1 h followed by 4-h chase (B). Homogenates prepared in the presence of nonionic detergent and low concentration of potassium were subjected to sedimentation velocity centrifugation on a continuous 5% to 25% (w/v) Suc gradient. Aliquots of gradient fractions, proportional amounts of the material precipitated at the bottom of the tubes (P), and an aliquot of total, nonfractionated homogenate (T) were subjected to immunoprecipitation with anti-GFP antiserum and analyzed by SDS-PAGE and radiography scanning. Top of gradients is at left, numbers on the top indicate the position along the gradient and the molecular mass of sedimentation markers, in kD. The asterisk indicates the 20-kD polypeptide that cofractionates with newly synthesized TPK1-GFP. Numbers at left indicate the positions of SDS-PAGE molecular mass markers, in kD.