Figure 3.

Phosphorylations of Nem1 and Spo7 by PKA are dependent on the amount of PKA, time of reaction, and concentration of ATP. Purified Nem1–Spo7 complex (0.35 pmol) was phosphorylated by PKA using [γ-³²P]ATP as described in the legend to Fig. 2. The PKA reaction was conducted by varying the amount of PKA (A), the reaction time (B), and the concentration of ATP (C). A and B, 40 μm ATP; B and C, 2.4 units PKA; A and C, 5 min. The amount of radioactive phosphate incorporated into Nem1 or Spo7 was determined from a standard curve using [γ-³²P]ATP. The amounts of Nem1 and Spo7 were determined by comparing their band intensities from a SYPRO Ruby–stained polyacrylamide gel with a standard curve of BSA. The positions of Nem1, Spo7, and molecular mass standards are indicated. The data shown in the plots are averages of three experiments ± S.D. (error bars). The portions of the phosphor images containing the phosphorylated Nem1 or Spo7 that are shown in the figure are representative of three experiments.