FIGURE 4.

Distinct Pγ domain preference of label transfer to α_tGTPγS and Pαβ competing for interaction with Pγ. A, label transfer from the [¹²⁵I]ACTP-Pγ photoprobes to α_tGTPγS in competition with Pαβ. [¹²⁵I]ACTP-Pγ photoprobes of the same batch as in Fig. 1 were used. Photocross-linking reactions were conducted with the [¹²⁵I]ACTP-Pγ photoprobes in the presence of both α_tGTPγS (4 μm) and Pαβ (without a nick) at a molar ratio of 0.7 Pγ, 1.0 α_t, 1.0 Pαβ subunit. The reactions were then subjected to SDS-PAGE (bottom) and autoradiography (top). BSA was used as an internal control to demonstrate the specificity of label transfer. B, labeling ratio of α_tGTPγS versus Pαβ. Each bar value represents a mean ± S.D. (error bars) (of 3–5 experiments). The amount of label transfer to α_tGTPγS or Pαβ from each Pγ position was normalized for the α_t (or Pαβ) protein amount and the specific activity of the corresponding [¹²⁵I]ACTP-Pγ probe (supplemental Table S1 and Ref. 28). A t test was performed for each position against the value 1.0 (dashed line): positions 40, 50, and 76 (***); positions 30 and 87 (**); positions 70 and 73 (*); and positions 21, 60, and 68 (not significant (ns)). A bar value less than 1 indicates a labeling preference for Pαβ; accordingly, a value greater than 1 indicates a preference for α_t.