Bipartite determinants mediate an evolutionarily conserved interaction between Cdc48 and the 20S peptidase

Abstract

Proteasomes are essential and ubiquitous ATP-dependent proteases that function in eukarya, archaea, and some bacteria. These destructive but critically important proteolytic machines use a 20S core peptidase and a hexameric ATPase associated with a variety of cellular activities (AAA+) unfolding ring that unfolds and spools substrates into the peptidase chamber. In archaea, 20S can function with the AAA+ Cdc48 or proteasome-activating nucleotidase (PAN) unfoldases. Both interactions are stabilized by C-terminal tripeptides in AAA+ subunits that dock into pockets on the 20S periphery. Here, we provide evidence that archaeal Cdc48 also uses a distinct set of near-axial interactions to bind 20S and propose that similar dual determinants mediate PAN-20S interactions and Rpt(1-6)-20S interactions in the 26S proteasome. Current dogma holds that the Rpt(1-6) unfolding ring of the 19S regulatory particle is the only AAA+ partner of eukaryotic 20S. By contrast, we show that mammalian Cdc48, a key player in cell-cycle regulation, membrane fusion, and endoplasmic-reticulum-associated degradation, activates mammalian 20S and find that a mouse Cdc48 variant supports protein degradation in combination with 20S. Our results suggest that eukaryotic Cdc48 orthologs function directly with 20S to maintain intracellular protein quality control.

Fig. 1.

Model for bipartite interactions between Cdc48 and 20S. (A) Domain architectures of the Cdc48, PAN, Mpa, and 19S Rpt AAA+ ATPases. Cdc48 consists of two highly homologous AAA+ domains (D1 and D2), a family-specific N-terminal domain, and a flexible and unstructured C-terminal tail consisting of a linker and the HbYX motif. Different family-specific N-domains and flexible unstructured C-termini terminating with an HbYX tripeptide are also found in the AAA+ single-ring unfoldases Mpa, PAN, and some 19S Rpt subunits. (B) Cartoon model of the archaeal Cdc48-20S proteasome. The N domains of Cdc48 are omitted for clarity. The HbYX tripeptides of Cdc48 dock into conserved binding pockets on the periphery of the 20S α ring. Near-axial interactions mediated by the pore-2 loops of the D2 ring of Cdc48 and the N-terminal gating residues of 20S also appear possible. (C) Upper, a WebLogo (28) representation of the C-terminal HbYX motif from archaeal and eukaryotic Cdc48. Lower, the sequence of pore-2 residues following the D2-ring Walker-B motif in archaeal and eukaryotic Cdc48. For functional studies, we deleted the central part of the loop (red) in archaeal Cdc48.

Fig. 2.

AAA+ D2 pore loops contribute to 20S binding. (A) Nonapeptide cleavage by ta20S peptidase (10 nM) as a function of increasing concentrations of taCdc48 variants. Solid lines are fits to a quadratic equation for near-stoichiometric binding. Values are averages (n = 3) ± SEM. Fitted K_app values for taCdc48^ΔN, taCdc48^ΔN/ΔC3, and taCdc48^ΔN/ΔC20 were 2 ± 0.5, 24 ± 4, and 42 ± 8 nM, respectively. The inset shows an SDS/PAGE assay of GFP-ssrA (5 µM) degradation by 1.2 µM taCdc48^ΔN/ΔC20 and 0.4 µM ta20S. Reactions were performed in the presence of 2 mM ATP and an ATP regeneration system or in the presence of 2 mM ATPγS. (B) ATP-hydrolysis rates, GFP-ssrA unfolding rates, and GFP-ssrA degradation rates are plotted for taCdc48 variants relative to these activities for taCdc48^ΔN. ATP hydrolysis was measured at 45 °C in the presence of an ATP regeneration system and 2 mM ATP. Unfolding of GFP-ssrA (5 µM) by taCdc48^ΔN variants (0.3 µM) was assayed at 60 °C in the presence of 10 mM ATP. Degradation of GFP-ssrA (5 µM) by taCdc48^ΔN variants (0.3 µM) and ta20S (0.9 µM) was measured at 45 °C in the presence of 5 mM ATP and an ATP regeneration system. Values are plotted as averages (n = 3) ± SEM. (C) Nonapeptide cleavage by ta20S as a function of increasing concentrations of taCdc48^∆N variants. Experiments were performed and analyzed as described in (A). For taCdc48^{ΔN/∆580–583}, the fitted K_app was 24 ± 4 nM. (D) Degradation of GFP-ssrA (5 µM) at 45 °C was assayed by changes in fluorescence in the presence of taCdc48^ΔN (50 nM) and increasing concentrations of ta20S or ta20S^∆α2–12. Values are averages (n = 3) ± SEM. (E) Nonapeptide cleavage by ta20S (10 nM) was assayed at 45 °C as a function of increasing concentrations of mjPAN or mjPAN^∆C3 in the presence of 100 µM ATPγS. Solid lines are fits to a quadratic equation for near-stoichiometric binding for mjPAN (K_app 54 ± 7 nM) and to a hyperbolic equation for mjPAN^∆C3 (K_app 3.8 ± 1 µM). Values are averages (n = 3) ± SEM.

Fig. 3.

Evolutionary conservation of a direct Cdc48–20S interaction. (A) Nonapeptide cleavage by the S. cerevisiae 20S peptidase (10 nM) as a function of increasing concentrations of taCdc48 or taCdc48^∆N. Experiments were performed at 37 °C in the presence of 2 mM ATP. Data were fit to a hyperbolic equation for taCdc48 (K_app 2.2 ± 0.2 µM) or a quadratic equation for near stoichiometric binding for taCdc48^∆N (K_app 24 ± 2 nM). Values are averages (n = 3) ± SEM. (B) SDS/PAGE assay of GFP-ssrA (5 µM) degradation by taCdc48^ΔN (1.2 µM) and sc20S (0.4 µM). Reactions were performed at 37 °C in the presence of 2 mM ATP and an ATP regeneration system. (C) Stimulation of nonapeptide cleavage by mouse 20S (5 nM) was assayed at 37 °C in the presence of 100 µM ATPγS and increasing concentrations of mouse Cdc48 or mouse Cdc48^∆N. The lines are fits to a hyperbolic equation with K_app values of 0.9 ± 0.2 µM (mmCdc48) and 1.0 ± 0.2 µM (mmCdc48^∆N). Values are averages (n = 3) ± SEM. (D) Stimulation of nonapeptide cleavage by archaeal ta20S peptidase (10 nM) by increasing concentrations of mouse Cdc48 or mouse Cdc48^∆N. The lines are fits to a quadratic equation for near stoichiometric binding with K_app values of 10 ± 2 nM (mmCdc48) and 5 ± 2 nM (mmCdc48^ΔN). Experiments were performed at 37 °C in the presence of ATP (2 mM).

Fig. 4.

Protein degradation by mouse 20S and a mouse Cdc48 variant. (A) In the mmCdc48^YY/ΔN variant of mouse Cdc48, the pore-1 loop of the D1 ring is mutated from KLAG to KYYG to match the sequence in taCdc48. The gel shows an SDS/PAGE assay of degradation of GFP-ssrA (5 µM) by mmCdc48^YY/ΔN (1.2 µM) and mm20S (0.4 µM) at 37 °C with 2 mM ATP and a pyruvate-kinase based ATP regeneration system. Degradation was inhibited by MG132 (100 µM). (B) SDS/PAGE assay of GFP-ssrA degradation by mmCdc48^YY/ΔN and ta20S using the same conditions as in (A). The gel strips on the bottom show that GFP-ssrA is not degraded without ATP or without mmCdc48^YY/ΔN. (C) Michaelis-Menten plots of the steady-state degradation of different concentrations of GFP-ssrA by ta20S (0.9 µM) and mmCdc48^YY/ΔN (0.3 µM). The fitted K_M was 1.7 ± 0.1 µM and V_max was 0.43 ± 0.01 min⁻¹ Cdc48₆⁻¹. (D) As assayed by loss of native fluorescence, GFP-ssrA (5 µM) was not degraded by ta20S (0.4 µM) in the presence of mmCdc48, mmCdc48^∆N, or mmCdc48^YY (1.2 µM) but was degraded when mmCdc48^YY/∆N (1.2 µM) was present.