An ALS disease mutation in Cdc48/p97 impairs 20S proteasome binding and proteolytic communication

Abstract

Cdc48 (also known as p97 or VCP) is an essential and highly abundant, double-ring AAA+ ATPase, which is ubiquitous in archaea and eukaryotes. In archaea, Cdc48 ring hexamers play a direct role in quality control by unfolding and translocating protein substrates into the degradation chamber of the 20S proteasome. Whether Cdc48 and 20S cooperate directly in protein degradation in eukaryotic cells is unclear. Two regions of Cdc48 are important for 20S binding, the pore-2 loop at the bottom of the D2 AAA+ ring and a C-terminal tripeptide. Here, we identify an aspartic acid in the pore-2 loop as an important element in 20S recognition. Importantly, mutation of this aspartate in human Cdc48 has been linked to familial amyotrophic lateral sclerosis (ALS). In archaeal or human Cdc48 variants, we find that mutation of this pore-2 residue impairs 20S binding and proteolytic communication but does not affect the stability of the hexamer or rates of ATP hydrolysis and protein unfolding. These results suggest that human Cdc48 interacts functionally with the 20S proteasome.

Keywords: AAA+ machine; ATP-dependent degradation; proteasome; protein unfolding.

Figure 1

Cdc48 and the 20S proteasome. A: Model for the T. acidophilum Cdc48^ΔN•20S proteasome based on the 3CF1 and 1PMA crystal structures and a low-resolution structure determined by electron microscopy. The red spheres mark the position of Asp⁵⁹² in human Cdc48, corresponding to Asp⁵⁸¹ in T. acidophilum Cdc48. B: In T. acidophilum and humans, the Cdc48 enzyme consists of a N-terminal domain, D1 and D2 AAA+ domains that mediate ATP hydrolysis and machine function, and a C-terminal tail with a terminal HbYX motif that stabilizes binding to the 20S proteasome. The positions and sequences of the pore-2 loop in the D2 ring of T. acidophilum and human Cdc48 are shown in red. Sequence conservation within this loop in archaeal and eukaryotic Cdc48 orthologs is shown in WebLogo representation.

Figure 2

Effects of pore-2 loop mutations in T. acidophilum Cdc48 variants lacking the N domain and HbYX tails. A: Rates of hydrolysis of ATP (5 mM) by T. acidophilum Cdc48 variants (0.3 µM) were measured at 45°C with ATP regeneration. B: Rates of Kaede-ssrA (10 µM) unfolding by taCdc48 variants (0.3 µM) were measured at 45°C with 5 mM ATP and ATP regeneration. C: Rates of ^SFGFP-ssrA (10 µM) degradation by Cdc48 variants (0.3 µM) and the T. acidophilum 20S proteasome (5 µM) were measured at 45°C with 5 mM ATP and ATP regeneration. D: Rates of ^SFGFP-ssrA (10 µM) degradation were measured as a function of increasing ta20S proteasome concentration in the presence of taCdc48 variants (50 nM) with 5 mM ATP and ATP regeneration. Lines are fits to the Hill equation. E: Rates of nonapeptide (10 µM) cleavage by the ta20S proteasome (10 nM) were assayed in the presence of increasing concentrations of taCdc48 variants with 2 mM ATP at 45°C. The lines are fits to a quadratic equation for near-stoichiometric binding. In all panels, values are plotted as averages (n ≥ 3) ± SEM and the parental enzyme is taCdc48^ΔN/ΔC20.

Figure 3

Archaeal D581 mutants show impaired 20S-proteasome binding and proteolytic communication in the presence of the HbYX tails. A: Rates of hydrolysis of ATP (5 mM) by taCdc48^ΔN variants (0.3 µM) were measured at 45°C. B: Rates of unfolding of Kaede-ssrA (10 µM) by taCdc48^ΔN variants (0.3 µM) with 5 mM ATP and ATP regeneration. C: Rates of ^SFGFP-ssrA (10 µM) degradation by taCdc48^ΔN variants (0.3 µM) and the ta20S proteasome (5 µM) at 45°C with 5 mM ATP and ATP regeneration. D: Rates of ^SFGFP-ssrA (10 µM) degradation by taCdc48^ΔN variants (50 nM) and increasing concentrations of the ta20S proteasome with 5 mM ATP and ATP regeneration. Lines are fits to the Hill equation. In all panels, values are plotted as averages (n ≥ 3) ± SEM.

Figure 4

Effects of the ALS-linked D592N mutation on the enzymatic activities of human Cd48^ΔN/YY. A: Rates of hydrolysis of ATP (5 mM) by hsCdc48^ΔN/YY variants (1 µM) were measured at 37°C. B: Rates of unfolding of Kaede-ssrA (10 µM) by hsCdc48^ΔN/YY variants (1 µM) were measured at 37°C in the presence of 5 mM ATP and ATP regeneration. C: Rates of ^SFGFP-ssrA (10 µM) degradation by hsCdc48^ΔN/YY variants (1 µM) and the human 20S proteasome (0.5 µM) were measured at 37°C with 5 mM ATP and ATP regeneration. In all panels, values are plotted as averages (n ≥ 3) ± SEM.