Positive cooperativity of the p97 AAA ATPase is critical for essential functions

Abstract

p97 is composed of two conserved AAA (ATPases associated with diverse cellular activities) domains, which form a tandem hexameric ring. We characterized the ATP hydrolysis mechanism of CDC-48.1, a p97 homolog of Caenorhabditis elegans. The ATPase activity of the N-terminal AAA domain was very low at physiological temperature, whereas the C-terminal AAA domain showed high ATPase activity in a coordinated fashion with positive cooperativity. The cooperativity and coordination are generated by different mechanisms because a noncooperative mutant still showed the coordination. Interestingly, the growth speed of yeast cells strongly related to the positive cooperativity rather than the ATPase activity itself, suggesting that the positive cooperativity is critical for the essential functions of p97.

FIGURE 1.

Structure of p97. A, schematic representation of CDC-48.1 from C. elegans and Cdc48p from S. cerevisiae. Mutation sites in the Walker A (A) and B (B) motifs, and Sensor-1 and arginine finger in the SRH are shown. The N-terminal domain of yeast Cdc48p is longer than CDC-48.1 by four residues. B, the crystal structure of the active ATPase site of p97 D1 showing ADP bound at the interface of two protomers. The Walker A motif (yellow), the Walker B motif (blue), and the SRH (red) are shown. Residues involving ATP binding and hydrolysis are shown (sticks). Lys of the Walker A motif, Glu of the Walker B motif, and Asn of the SRH work in cis, whereas Arg of the SRH works in trans.

FIGURE 2.

Characterization of the ATPase activity of CDC-48.1 mutants. ATPase activity was measured at 30 °C by the malachite green assay. The ATPase activity of wild type CDC-48.1 was set to 100%. Error bars represent the standard deviations from three independent experiments.

FIGURE 3.

Kinetic analysis of the ATPase activity of CDC-48.1 mutants. ATP hydrolysis rates of CDC-48.1^WT (A), CDC-48.1^E311Q (B), CDC-48.1^E311Q/N354A (C), and CDC-48.1^E311Q/R365A (D) in the presence of the indicated concentrations of ATP and magnesium chloride were measured at room temperature using the enzyme-coupled assay. The solid lines were obtained by a least-squares fit to the equation 1 as described under “Experimental Procedures,” and the kinetic parameters were calculated. The dashed lines are fitting curves when n_H is fixed to 1.

FIGURE 4.

Effects of D1 Walker B mutations on the growth of S. cerevisiae. Yeast cells expressing wild type Cdc48p and mutant forms of Cdc48p as a sole Cdc48 copy were diluted in 10-fold increments, and 5 μl of each dilution (starting with the 0.001 A₆₀₀ unit) were spotted onto SD medium lacking histidine. Cells were grown at 25 °C and 37 °C.

FIGURE 5.

Relationships between the yeast growth rate and Hill coefficient or ATPase activity. Yeast cells were grown in liquid SD medium at 37 °C, and the growth was monitored by measuring A₆₀₀, and the growth rates were determined from log-phase growth. The growth rate of mutant cells was plotted against the ATPase activity (A) and the Hill coefficient (B) of the corresponding CDC-48.1 mutants. The correlation coefficient (r) was determined by fitting the data as a linear model.

FIGURE 6.

Inhibition of ATPase activity by ATPγS. ATP hydrolysis by CDC-48.1 mutants and ClpX in the presence of indicated amounts of ATPγS with the constant concentration of ATP (3 mm) was measured at 30 °C by the malachite green assay. The ATPase activity of each protein in the absence of ATPγS was set to 100%.

FIGURE 7.

Proposed mechanisms of ATP hydrolysis by p97. See details in the text.