Allosteric activation of Sac1 by its product PtdIns. (A) Concentration-dependent activation of Sac1 by PtdIns. The reactions were conducted with a fixed amount of substrate PI(4)P and varying amounts of PtdIns. Initial velocities obtained from each progress curve were plotted vs PtdIns concentration. (B) PI(4)P hydrolysis in the presence of 50 μM diC₈ PtdIns (●). The initial velocity data were plotted and fit to the Michaelis–Menten equation. Full activation of Sac1 by PtdIns results in a hyperbolic kinetic curve. For comparison, the kinetic curve without an activator (▽, the same curve shown in Figure 1A) is also plotted on the same scale. (C) Sedimentation velocity AUC measurements of Sac1 in solution. Differential sedimentation coefficient distribution c(s) plotted vs sedimentation coefficient. Experiments were conducted with a protein concentration of 0.1 mg/mL in the absence of PtdIns (—) or in the presence of 20 (−–−) or 50 μM diC₈ PtdIns (···).

Hydrolysis of diC₁₆ PI(4)P substrates presented in liposomes by Sac1. (A) Representative reaction progress curves of the hydrolysis of diC₁₆ PI(4)P in liposomes with different lipid compositions by Sac1. (B) Activity of Sac1 toward liposomes with different lipid compositions (n = 2). The initial velocity data were extracted from progress curves shown in panel A. (C) Reaction progress curves of the hydrolysis of diC₁₆ PI(4)P in liposomes with a fixed amount of PI(4)P substrate but with a different molar fraction of PS. (D) Initial velocities obtained from panel C plotted vs the mole fraction of PS.

Sac1 is an allosteric enzyme. (A) Concentration dependence of PI(4)P hydrolysis. Initial velocities were determined from reaction progress curves and were plotted vs substrate concentration (x-axis). The data were fit to the Hill equation. The Hill coefficient h is 4.5 ± 0.3, and the K_0.5 is 16 ± 2 μM. (B) Sedimentation velocity AUC measurements of Sac1 in solution at different concentrations [1 (—) and 0.1 mg/mL (---)]. The differential sedimentation coefficient distribution c(s) is plotted vs the sedimentation coefficient. Sac1 is predominantly in the monomeric form (95%) at a protein concentration of 0.1 mg/mL (---).

Sac1 mutants exhibit altered kinetics. (A) Surface representative of Sac1 phosphatase. The surfaces were colored on the basis of electrostatic potential with positively charged regions colored blue (+3 kcal/electron) and negatively charged surfaces red (−3 kcal/electron). Sac1 has a deep cationic cleft where the catalytic site resides (left). The catalytic P-loop with the unique conformation is shown as a red tube. Residues selected for mutagenesis are labeled and shown as sticks (close-up on the right). (B) Plot of the kinetics of the R207Q mutant. The initial velocities were plotted vs substrate concentration. Fitting with the Hill equation revealed a significant increase in the K_0.5 to ∼46 μM. (C) Hydrolysis of PI(4)P by the Sac1 R207Q mutant in the presence of 20 μM diC₈ PS. The initial velocity data were plotted and fit to the Michaelis–Menten equation. (D) Schematic diagram for the allosteric activation of Sac1. When allosteric activators such as PtdIns or PS bind, the catalytic P-loop may change its conformation, allowing substrate binding and active catalysis by Sac1.

Elevated PI(4)P levels in cho1Δ yeast cells. (A) Metabolic pathway of PS. Cho1 is a phosphatidylserine synthase that catalyzes the synthesis of PS from CDP-diaclyglycerol and l-serine. PS can be used to generate PE. CHO1-deficient yeast cells are viable, provided the growth medium is supplemented with ethanolamine that can be used to synthesize PE through the Kennedy pathway.⁴⁵(B) PI(4)P localization in wild-type and cho1Δ cells. Cells expressing mCherry-2xPH^Osh2 were observed by fluorescence microscopy. Mother cells are indicated (m). The scale bar is 5 μm. (C) Cellular PI(4)P levels in wild-type and cho1Δ cells were measured by [³H]-myo-inositol labeling. Lipids from wild-type (gray) and cho1Δ (black) cells were extracted and deacylated for analysis by HPLC. Error bars denote the standard deviation of three experiments.