Journal of Structural Biology 156 (2006) 72–83 www.elsevier.com/locate/yjsbi Review Proteasomes and their associated ATPases: A destructive combination David M. Smith a, Nadia Benaroudj b, Alfred Goldberg a,¤ a Harvard Medical School, Department of Cell Biology, 240 Longwood Avenue, Boston, MA 02115, USA b Pasteur Institute, Unit of Bacterial Genetics and DiVerentiation, 25-28 rue du Dr. Roux, 75724 Paris Cedex 15, France Received 24 February 2006; received in revised form 19 April 2006; accepted 19 April 2006 Available online 8 May 2006 Abstract Protein degradation by 20S proteasomes in vivo requires ATP hydrolysis by associated hexameric AAA ATPase complexes such as PAN in archaea and the homologous ATPases in the eukaryotic 26S proteasome. This review discusses recent insights into their multistep mechanisms and the roles of ATP. We have focused on the PAN complex, which oVers many advantages for mechanistic and structural studies over the more complex 26S proteasome. By single-particle EM, PAN resembles a “top-hat” capping the ends of the 20S protea- some and resembles densities in the base of the 19S regulatory complex. The binding of ATP promotes formation of the PAN–20S com- plex, which induces opening of a gate for substrate entry into the 20S. PAN’s C-termini, containing a conserved motif, docks into pockets in the 20S’s ring and causes gate opening. Surprisingly, once substrates are unfolded, their translocation into the 20S requires ATP- binding but not hydrolysis and can occur by facilitated diVusion through the ATPase in its ATP-bound form. ATP therefore serves multiple functions in proteolysis and the only step that absolutely requires ATP hydrolysis is the unfolding of globular proteins. The 26S proteasome appears to function by similar mechanisms. © 2006 Elsevier Inc. All rights reserved. Keywords: Proteasome; ATPase; PAN; Proteasome activating nucleotidase; 20S; 26S; RPT; 19S; HbYX; Gate; YDR; ATP; ATPS; Translocation; Protein; Brownian ratchet; Brownian; Proteasomal ATPases; Archaea; EM; Electron microscopy; LFP; Degradation; Protein degradation; Turnover 1. Introduction teins and ATP in linked processes (Chung and Goldberg, 1981; Gottesman, 1996). A fundamental feature of protein breakdown in eukary- In eukaryotes, ATP is required both for ubiquitin conju- otic and prokaryotic cells is its requirement for ATP (Gold- gation to substrates and for the function of the 26S protea- berg and St. John, 1976). Much of our current knowledge some, the ATP-dependent complex that catalyzes the about intracellular proteolysis came from studies seeking to breakdown of ubiquitinated and certain non-ubiquitinated understand the biochemical basis of this surprising require- polypeptides (Ciechanover, 2005; Goldberg, 2005; Voges ment (Ciechanover, 2005; Goldberg, 2005). The key early et al., 1999). The discovery of the Wrst ATP-dependent pro- developments were the discovery of a soluble (nonlysoso- tease in bacteria (lon/La) (Chung and Goldberg, 1981) was mal) ATP-dependent proteolytic system in reticulocytes made about the same time as the classic discovery of the (Etlinger and Goldberg, 1977) followed by the establish- role of ubiquitin in protein breakdown in the reticulocyte ment of similar energy-dependent proteolytic systems in system by Hershko, Ciechanover, and Rose (Ciechanover, extracts of Escherichia coli (Murakami et al., 1979). Analy- 2005; Glickman and Ciechanover, 2002). The energy- sis of these bacterial systems led to the discovery of large requirement for ubiquitin conjugation in eukaryotes was ATP-dependent proteolytic complexes that degrade pro- thought to explain the ATP requirements for intracellular proteolysis in eukaryotes. Thus, initially it was believed that there are two very diVerent explanations for the ATP * Corresponding author. requirements for proteolysis in prokaryotes and eukary- E-mail address: [email protected] (A. Goldberg). otes. However, after further study, it became clear that after 1047-8477/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2006.04.012 D.M. Smith et al. / Journal of Structural Biology 156 (2006) 72–83 73 ubiquitination, ATP was still required for breakdown of the AAA+ (ATPases Associated with various cellular the protein (Tanaka et al., 1983), and by the late 1980s, the Activities) ATPase superfamily (for review see Ogura and 26S proteasome was identiWed as the ATP-dependent pro- Tanaka, 2003). The AAA+ family of ATPases are found in teolytic complex that degrades ubiquitinated proteins all living organisms and in all cell compartments, where (Hough et al., 1987; Waxman et al., 1987). they participate in a variety of essential cellular processes As interest in the eukaryotic 20S proteasome developed, such as mitosis, protein folding and translocation, DNA archaea were found to contain a simpler, but structurally replication and repair, membrane fusion and proteolysis. markedly similar proteolytic complex in Thermoplasma aci- They are characterized by the presence of one or two con- dophilum (Baumeister et al., 1998; Dahlmann et al., 1989; served ATP-binding domains (200–250 residues), called the Voges et al., 1999). Further work also uncovered the exis- AAA motif, consisting of a Walker A and a Walker B motif tence of an ATPase complex, PAN, which functions (Confalonieri and Duguet, 1995). The eukaryotic and together with the archaeal 20S proteasome (Benaroudj and archaeal (PAN) proteasomal ATPases belong to a subfam- Goldberg, 2000; Smith et al., 2005; Zwickl et al., 1999). ily of AAA+ ATPases (AAA family) that contains an addi- Thus, protein breakdown in archaea, bacteria and eukary- tional motif called the second region of homology (SRH) otes is catalyzed by large proteolytic complexes that hydro- (Lupas and Martin, 2002). Despite the large variety of cel- lyze ATP and protein in linked reactions. Interestingly, lular processes in which AAA+ ATPases participate, they PAN is not found in all archaea (e.g., T. acidophilum). How- have some common features. A recurrent structural feature ever, it appears likely that all archaea contain ATPase ring of most AAA+ ATPases is their assembly into oligomeric complexes of the AAA family that may also function in (generally hexameric) ring-shaped structures with a central protein degradation by the proteasome. For example, VAT, pore. In addition, most appear to be involved in protein which is found in T. acidophilum, seems likely to function in folding or unfolding, and assembly or disassembly of pro- substrate recognition, unfolding, and translocation of sub- tein complexes through nucleotide-dependent conforma- strates into the 20S proteasome (Gerega et al., 2005). tional changes. Thus, recent insights into the functioning of the archaeal PAN complex and the 19S proteasomal regu- 1.1. The 26S proteasome latory ATPase may illuminate the functioning of these other AAA Family members (and vise versa). The ATP-dependent 26S proteasome is composed of one Though bacteria do not contain 20S proteasomes, like or two 19S regulatory complexes and the central 20S particle those in eukaryotes, they do contain several large compart- (Voges et al., 1999; Zwickl et al., 1999), which is a hollow cyl- mentalized protease complexes that associate with AAA inder, within which proteolysis occurs. The two outer rings ATPase complexes such as HslUV and ClpAP. HslV is a and two inner rings of the 20S particles are each composed two-ring peptidase complex which shares homology with of seven distinct but homologous subunits. In eukaryotes, the beta subunits of the 20S proteasome (Bochtler et al., three of the subunits contain proteolytic sites, which are 2000), and forms a six-membered ring (Rohrwild et al., sequestered in the hollow interior of the 20S particle (Groll 1997) rather than the seven-membered ring, which is char- et al., 1997). Substrates enter the 20S through a narrow chan- acteristic of the 20S proteasome. HslU, the ATPase com- nel formed by the subunits, whose N-termini, depending on plex, associates with HslV to stimulate protein degradation, their conformation, can either obstruct or allow substrate and is homologous to PAN. X-ray diVraction studies estab- entry and thus function as a gate (Groll et al., 2000; Groll lished that HslU induces conformational changes in the and Huber, 2003). This entry channel is narrow and only per- peptidase active site of HslV upon association and mits passage of unfolded, linearized polypeptides (Groll increases the pore size of HslV. Thus, HslU increases the et al., 1997). The 19S regulatory complex is composed of two peptidase activity of HslV by allosteric activation and subcomplexes, the lid, which seems to bind and disassemble probably also by promoting substrate unfolding for peptide the ubiquitin-conjugated substrate, and the base, which con- entry (Huang and Goldberg, 1997; Sousa et al., 2000; Wang tains six homologous ATPase subunits (termed Rpt1–6 in et al., 2001; Yoo et al., 1997). Facilitating peptide entry thus yeast) plus two non-ATPases, Rpn 1 and 2 (Voges et al., appears to be a common property shared by HslU, PAN, 1999). These ATPases are members of the AAA family of and the 19S ATPases, although HslV does not contain an ATPases (Patel and Latterich, 1998). For a globular protein outer ring or gating termini like those in the 20S protea- to be degraded, it must associate with the 19S ATPases and some. undergo ATP-dependent unfolding followed by transloca- tion into the 20S particle, which requires opening of the gate 2. The function of the 26S ATPases in the ring (Kohler et al., 2001). Each of these steps is regu- lated in some way by the ATPase complex. Studying the ATP-dependent processes and the mecha- nisms of protein breakdown within the 26S proteasome has 1.2. AAA ATPases and proteolysis proven diYcult because of its structural complexity, multi- ple enzymatic activities and ubiquitin requirement. Never- The ATPase complexes that regulate protein degrada- theless, several important discoveries about these ATPases tion in eukaryotes, bacteria and archaea are all members of have been made using genetic tools in yeast.