Figure 2. Degradation of PdeA Couples a Naturally Weak C-Terminal Degron with a Distal Tethering Domain.

(A) A GFP reporter fusion containing only the C-terminal residues of PdeA (GFP-9PdeA) is degraded more slowly than the full-length PdeA (GFP-PdeA), but is degraded faster than GFP-9PdeADD, which contains a carboxyl-terminal Asp-Asp. Representative curves are of 1 μM substrates in the presence of 2.5 μM CpdR using standard conditions.

(B) Degradation of full-length PdeA without CpdR is inefficient but is substantially enhanced in the presence of CpdR while GFP-9PdeA degradation is unaffected by CpdR. Inset highlights the more slowly degraded substrates.

(C) CpdR can preferentially improve the K_M of a readily degraded substrate. PdeA constructs containing a strong recognition tag (GFP-PAS-ssrA) show an increase in K_M upon addition of CpdR, with little change in v_max relative to the degradation of this substrate in the absence of CpdR. Replacing the ssrA tag with a weaker tag also shows an improvement of K_M in the presence of CpdR (Figure S2). In B and C, lines are fits to the Michaelis-Menten equation; parameters are shown in Table 1.