Identification of the Cdc48•20S Proteasome as an Ancient AAA+ Proteolytic Machine

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Science  17 Aug 2012:
Vol. 337, Issue 6096, pp. 843-846
DOI: 10.1126/science.1224352


Proteasomes are the major energy-dependent proteolytic machines in the eukaryotic and archaeal domains of life. To execute protein degradation, the 20S core peptidase combines with the AAA+ ring of the 19S regulatory particle in eukarya or with the AAA+ proteasome-activating nucleotidase ring in some archaea. Here, we find that Cdc48 and 20S from the archaeon Thermoplasma acidophilum interact to form a functional proteasome. Cdc48 is an abundant and essential double-ring AAA+ molecular machine ubiquitously present in archaea, where its function has been uncertain, and in eukarya where Cdc48 participates by largely unknown mechanisms in diverse cellular processes, including multiple proteolytic pathways. Thus, proteolysis in collaboration with the 20S peptidase may represent an ancestral function of the Cdc48 family.

In all domains of life, protein-unfolding machines fueled by adenosine triphosphate (ATP) and self-compartmentalized peptidases carry out intracellular protein degradation (1). The unfoldases belong to the AAA+ superfamily [adenosine triphosphatases (ATPases) associated with a variety of cellular activities] and function as hexameric rings. The barrel-shaped peptidases have an internal chamber containing the proteolytic active sites. All AAA+ proteases share common operating principles, including coaxial unfoldase-peptidase docking and translocation of unfolded substrates through the axial pores into the degradation chamber. The major degradation machine in eukaryotes is the 26S proteasome, consisting of the 20S core peptidase and 19S regulatory particles, which contain AAA+ Rpt1-6 unfolding rings (2, 3). The 20S peptidase, which has an α7β7β7α7 architecture, is also ubiquitous in archaea, where it functions with the hexameric AAA+ proteasome-activating nucleotidase (PAN) (4). Paradoxically, PAN is absent from some archaea (table S1) and is not required for viability of other archaea in which 20S is essential (5). Thus, we reasoned that an unidentified pathway for proteasomal protein degradation must exist.

Cdc48/p97 is a double-ring AAA+ machine (6) that is highly conserved but has no clear function in archaea. By contrast, eukaryotic Cdc48 and specific adaptor proteins participate in diverse cellular processes, including proteolytic pathways, but by largely unknown molecular mechanisms (6). It is intriguing that many archaeal and eukaryotic Cdc48 enzymes have C-terminal HbYX (hydrophobic residue, tyrosine, any residue) motifs, which are also present in PAN and Rpt1-6 subunits and dock into binding pockets on the 20S α ring (7).

The single AAA+ module of PAN is highly homologous to both AAA+ modules of Cdc48, but these proteins have distinct N domains (Fig. 1A) (8, 9). Searches of complete archaeal genomes revealed that ~15% contained no PAN genes but did contain 20S genes and at least one Cdc48 gene encoding an HbYX or related C-terminal sequence (Fig. 1B and table S1). Docking the crystal structures of Thermoplasma acidophilum 20S and mouse Cdc48/p97 showed that the C-terminal tails could be positioned to interact with the HbYX-binding clefts in the 20S α ring (fig. S1).

Fig. 1

Cdc48 binds the 20S peptidase. (A) PAN and Cdc48 have distinct N domains but homologous AAA+ modules and HbYX motifs. (B) Examples of archaea in which PAN was absent but 20S and Cdc48 were present. Red Cdc48 tripeptides match HbYX or related sequences. (C) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that purified T. acidophilum His6-Cdc48EQ/EQ pulled down two proteins from a T. acidophilum cell extract, which were identified as the 20S α and β subunits (fig. S2). (D) Using purified T. acidophilum proteins, 20S with a His6-tagged β subunit pulled down a roughly stoichiometric quantity of untagged Cdc48EQ/EQ in the presence of 2 mM ATP but not ADP.

For interaction studies, we used T. acidophilum His6-Cdc48EQ/EQ, a hexahistidine-labeled variant with E291Q/E568Q substitutions (glutamine for glutamic acid at positions 291 and 568) in the Walker-B motifs of the D1 and D2 rings to prevent ATP hydrolysis. When His6-Cdc48EQ/EQ was added to a T. acidophilum cell extract, it pulled down two proteins (Fig. 1C), identified as the 20S α and β subunits (fig. S2). Using purified His6-tagged 20S and purified untagged Cdc48EQ/EQ, we also observed a direct and approximately stoichiometric interaction in the presence of ATP but not adenosine diphosphate (ADP) (Fig. 1E).

The N termini of the α subunits gate or restrict entry of peptides longer than five to seven residues into free 20S, and such peptides are cleaved at enhanced rates by PAN•20S or the 26S proteasome (10). With ATP present, Cdc48 also had gate-opening activity, stimulating 20S cleavage of a nonapeptide but not a tetrapeptide (Fig. 2A and fig. S3). When the gate residues were deleted (10), 20SΔα2-12 alone cleaved the nonapeptide at a rate similar to that of Cdc48•20S, and added Cdc48 did not increase 20SΔα2-12 activity (Fig. 2B). Cdc48∆N, which lacks the N domain, stimulated nonapeptide cleavage in the presence of ATP and two poorly hydrolyzable analogs, ATPγS (adenosine 5′-[γ-thio]triphosphate and AMP-PNP (adenosine 5′-[β,γ-imido]triphosphate), whereas these analogs supported very weak gate opening by Cdc48 (Fig. 2B).

Fig. 2

Cdc48 activates 20S gate opening. (A) T. acidophilum Cdc48 (0.4 μM) and 20S (10 nM) cleaved a nonapeptide (10 μM) rapidly as assayed by increased fluorescence with 2 mM ATP. Cleavage by Cdc48 alone was undetected and by 20S alone was slow. (B) Cdc48 or Cdc48∆N (0.4 μM) stimulation of nonapeptide cleavage by 20S, 20S∆α2-12, or 20SαK66A (10 nM) in the presence of different nucleotides (2 mM). Values are averages ± SD (N ≥ 3) divided by the 20S cleavage rate. (C) Nonapeptide cleavage by 20S or 20SαK66A (10 nM) as a function of increasing concentrations of Cdc48 or variants. Solid lines (R2 ≥ 0.98) are fits to a hyperbolic equation for all curves except Cdc48∆N•20S, which was fit to a quadratic equation for near-stoichiometric binding. (D) Values for the maximum stimulation of cleavage and the apparent dissociation constant (Kapp) were calculated from the fits in (C) or fig. S4.

Apparent equilibrium dissociation constants of ~160 nM for Cdc48•20S and ~300 nM for PAN•20S were determined by measuring the Cdc48/PAN dependence of 20S nonapeptide cleavage (Fig. 2C and fig. S4). Cdc48∆N bound 20S substantially more tightly, with an affinity of ~2 nM (Fig. 2C).

A Cdc48 variant with the Y753A mutation (alanine replacing Tyr753) in the HbYX motif bound 20S and stimulated peptide cleavage (Fig. 2C) but with decreased affinity (~580 nM) and only 14% of the maximum activity of Cdc48 (Fig. 2D). Thus, binding and gate opening are not fully coupled in this mutant. In the binding pocket on 20Sα, Lys66 forms a salt bridge with the α-carboxylate of PAN (11). Cdc48 or Cdc48∆N stimulated nonapeptide cleavage by 20SαK66A poorly (Fig. 2, B and C), which suggests that interactions involving the Cdc48 tail and Lys66 facilitate gate opening.

T. acidophilum Cdc48 unfolds green fluorescent protein (GFP) bearing the ssrA degron, with unfolding and ATP hydrolysis being stimulated by 120 mM Mg++ or N-domain deletion (12). We found that Cdc48•20S completely degraded native GFP-ssrA in the presence of ATP and 120 mM Mg++ (Fig. 3A). Cdc48∆N•20S also efficiently degraded GFP-ssrA with ATP and 20 mM Mg++, but no degradation was observed with ATPγS, without Cdc48∆N, or with proteolytically inactive 20SβT1A (Fig. 3B). Michaelis-Menten analysis of GFP-ssrA degradation by Cdc48•20S showed that the Michaelis constant KM was ~2 μM in high and low Mg++, whereas the maximum initial velocity of the enzyme-catalyzed reaction, Vmax, increased from 0.07 to 0.39 min–1 enzyme–1 as Mg++ was raised (Fig. 3C and table S2). For Cdc48∆N•20S in low Mg++, KM was 0.15 μM and Vmax was 3.2 min–1 enzyme–1. Thus, the N domain of Cdc48 represses proteolytic activity both by increasing KM and decreasing Vmax. For comparison, PAN•20S degraded GFP-ssrA with a KM of 0.15 μM and Vmax of 0.35 min–1 enzyme–1 (Fig. 3C and table S2).

Fig. 3

Protein degradation by the Cdc48•20S proteasome. (A) SDS-PAGE assay of GFP-ssrA (10 μM) degradation by Cdc48 (1 μM) and 20S (2 μM) with 2 mM ATP, 120 mM MgCl2, and an ATP regeneration system. (B) (Top) SDS-PAGE assay of GFP-ssrA (5 μM) degradation by 20S (0.9 μM) and Cdc48∆N (0.3 μM), 2 mM ATP, 20 mM MgCl2, and a regeneration system. (Bottom strips) GFP-ssrA in otherwise comparable assays using 2 mM ATPγS (no regeneration), without Cdc48∆N, or with catalytically inactive 20SβT1A. (C) Michaelis-Menten plots of GFP-ssrA degradation by 20S (0.9 μM) and Cdc48 (0.3 μM; left) or 20S (0.9 μM) with Cdc48∆N or PAN (0.3 μM; right). Values are averages ± SD (N ≥ 3). Fitted parameters are listed in table S2. (D) Degradation of 10 μM native titinI27-ssrA or denatured CM-titinI27-ssrA. The top two gel strips show degradation by Cdc48 (1.2 μM) and 20S (0.4 μM) in 120 mM Mg++ buffer. The next four strips show degradation by Cdc48ΔN or Cdc48ΔN/EQ/EQ (1.2 μM) and 20S (0.4 μM) in 20 mM Mg++ buffer. The bottom strip shows degradation by 20S (0.4 μM) in 20 mM Mg++ buffer. Reactions contained 5 mM ATP and a regeneration system or 5 mM ATPγS without regeneration.

Cdc48•20S degraded CM-titinI27-ssrA, a variant denatured by carboxymethylation of normally buried cysteines (13), faster than native titinI27-ssrA (Fig. 3D). Cdc48∆N•20S also degraded the denatured substrate more rapidly, in a reaction that required ATP hydrolysis (Fig. 3D). In principle, Cdc48 might unfold and release substrates into solution for subsequent capture and degradation by free 20S. However, degradation of unfolded CM-titinI27-ssrA by 20S alone was much slower than degradation with Cdc48 or Cdc48∆N (Fig. 3D), which suggested that ATP-fueled translocation from Cdc48 into the associated 20S peptidase is responsible for most degradation. Moreover, a PAN variant that unfolded GFP-ssrA, but did not bind 20S, failed to support degradation (figs. S4 and S5). Thus, folded and unfolded substrates are only degraded efficiently by complexes of 20S with Cdc48 or PAN.

A ∆HbYX Cdc48∆N variant that terminates with Asp-Gln-Gly supported a low level of gate opening (Fig. 4A) and 20S degradation of GFP-ssrA (Fig. 4B). Although the affinity of Cdc48∆N/∆HbYX for 20S was reduced compared with that of Cdc48∆N, it was still surprisingly strong (40 nM). Because the HbYX motif stabilizes but is not required for 20S binding, other Cdc48 regions must make major contributions to binding. We also observed weak gate opening (Fig. 4A) and GFP-ssrA degradation (Fig. 4B) when we used 20SαK66A in combination with either Cdc48∆N or Cdc48∆N/∆HbYX, showing that K66 recognition of an α-carboxylate is not essential for binding and degradation. Moreover, the affinity effects of the Cdc48 ∆HbYX and 20S αK66A mutations were nonadditive, as expected for interacting groups. How can the protein degradation activities of Cdc48∆N/∆HbYX•20S, Cdc48∆N•20SαK66A, and Cdc48∆N/∆HbYX•20SαK66A, which require an open 20S gate, be reconciled with the poor stimulation of peptide cleavage by these enzymes? The 20S gate dynamically adopts a variety of conformations (14), and an actively translocating polypeptide might trap a transiently open gate with the spooling chain preventing closure.

Fig. 4

The HbYX motif is not required for 20S binding and degradation. (A) Stimulation of peptide cleavage by 20S or 20SαK66A (10 nM) as a function of Cdc48∆N/HbYX or Cdc48∆N concentration. Values are averages (N = 3) ± SD. Solid lines (R2 ≥ 0.948) are fits to a quadratic equation for near-stoichiometric binding, yielding Kapp values of 41 ± 4 nM for Cdc48∆N/HbYX•20S, 21 ± 9 nM for Cdc48∆N•20SαK66A, and 15 ± 6 nM for Cdc48∆N/HbYX•20SαK66A. (B) Gel strips showing ATP-dependent degradation of GFP-ssrA (5 μM) by Cdc48ΔN/∆HbYX•20S, Cdc48ΔN•20SαK66A, or Cdc48∆N/HbYX•20SαK66A. Assays were performed in 20 mM Mg++ using 5 mM ATP and regeneration or 5 mM ATPγS; 20S/20SαK66A (0.4 μM); Cdc48ΔN/Cdc48∆N/HbYX (1.2 μM).

We find that archaeal Cdc48 binds to the proteasomal 20S peptidase, induces gate opening, and unfolds and actively translocates protein substrates into the 20S chamber for proteolysis. Cdc48 is the only 20S partner in ~15% of archaea, and Cdc48 and PAN are both present in the remaining ~85%. On the basis of their C-terminal sequences (table S1), the fact that Cdc48 binding is not strictly HbYX-dependent, and our finding that Cdc48 and PAN bind 20S with similar affinities (Fig. 2D), it appears that either enzyme could combine with 20S to form alternative proteasomes in most archaea. The N domain of Cdc48 is a tethering site for adaptors and possibly substrates, and there is evidence that it controls hexamer conformation and ATP-hydrolysis rates (12, 1517). In our experiments, deleting the N domain of T. acidophilum Cdc48 strengthened 20S binding and increased proteolytic activity. Thus, N-domain modification or N-domain binding of substrates and adaptors may regulate intracellular degradation by the Cdc48•20S proteasome.

Supplementary Materials

Materials and Methods

Figs. S1 to S5

Tables S1 and S2

References (1823)

References and Notes

  1. Acknowledgments: We thank C. Hill, A. Horwich, H. Huber, S. Kim, K. Schmitz, B. Stinson, and M. Wohlever for materials and discussions. Supported by NIH grant AI-16892. The data reported in this manuscript are presented in the main paper and supplementary materials.
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