Review
Proteasomes and their associated ATPases: A destructive combination

https://doi.org/10.1016/j.jsb.2006.04.012Get rights and content

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 offers 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 proteasome and resembles densities in the base of the 19S regulatory complex. The binding of ATP promotes formation of the PAN–20S complex, 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 α 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 diffusion 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.

Introduction

A fundamental feature of protein breakdown in eukaryotic and prokaryotic cells is its requirement for ATP (Goldberg and St. John, 1976). Much of our current knowledge about intracellular proteolysis came from studies seeking to understand the biochemical basis of this surprising requirement (Ciechanover, 2005, Goldberg, 2005). The key early developments were the discovery of a soluble (nonlysosomal) ATP-dependent proteolytic system in reticulocytes (Etlinger and Goldberg, 1977) followed by the establishment of similar energy-dependent proteolytic systems in extracts of Escherichia coli (Murakami et al., 1979). Analysis of these bacterial systems led to the discovery of large ATP-dependent proteolytic complexes that degrade proteins and ATP in linked processes (Chung and Goldberg, 1981, Gottesman, 1996).

In eukaryotes, ATP is required both for ubiquitin conjugation to substrates and for the function of the 26S proteasome, the ATP-dependent complex that catalyzes the breakdown of ubiquitinated and certain non-ubiquitinated polypeptides (Ciechanover, 2005, Goldberg, 2005, Voges et al., 1999). The discovery of the first ATP-dependent protease in bacteria (lon/La) (Chung and Goldberg, 1981) was made about the same time as the classic discovery of the role of ubiquitin in protein breakdown in the reticulocyte system by Hershko, Ciechanover, and Rose (Ciechanover, 2005, Glickman and Ciechanover, 2002). The energy-requirement for ubiquitin conjugation in eukaryotes was thought to explain the ATP requirements for intracellular proteolysis in eukaryotes. Thus, initially it was believed that there are two very different explanations for the ATP requirements for proteolysis in prokaryotes and eukaryotes. However, after further study, it became clear that after ubiquitination, ATP was still required for breakdown of the protein (Tanaka et al., 1983), and by the late 1980s, the 26S proteasome was identified as the ATP-dependent proteolytic complex that degrades ubiquitinated proteins (Hough et al., 1987, Waxman et al., 1987).

As interest in the eukaryotic 20S proteasome developed, archaea were found to contain a simpler, but structurally markedly similar proteolytic complex in Thermoplasma acidophilum (Baumeister et al., 1998, Dahlmann et al., 1989, Voges et al., 1999). Further work also uncovered the existence of an ATPase complex, PAN, which functions together with the archaeal 20S proteasome (Benaroudj and Goldberg, 2000, Smith et al., 2005, Zwickl et al., 1999). Thus, protein breakdown in archaea, bacteria and eukaryotes is catalyzed by large proteolytic complexes that hydrolyze ATP and protein in linked reactions. Interestingly, PAN is not found in all archaea (e.g., T. acidophilum). However, it appears likely that all archaea contain ATPase ring complexes of the AAA family that may also function in protein degradation by the proteasome. For example, VAT, which is found in T. acidophilum, seems likely to function in substrate recognition, unfolding, and translocation of substrates into the 20S proteasome (Gerega et al., 2005).

The ATP-dependent 26S proteasome is composed of one or two 19S regulatory complexes and the central 20S particle (Voges et al., 1999, Zwickl et al., 1999), which is a hollow cylinder, within which proteolysis occurs. The two outer α rings and two inner β rings of the 20S particles are each composed of seven distinct but homologous subunits. In eukaryotes, three of the β subunits contain proteolytic sites, which are sequestered in the hollow interior of the 20S particle (Groll et al., 1997). Substrates enter the 20S through a narrow channel formed by the α subunits, whose N-termini, depending on their conformation, can either obstruct or allow substrate entry and thus function as a gate (Groll et al., 2000, Groll and Huber, 2003). This entry channel is narrow and only permits passage of unfolded, linearized polypeptides (Groll et al., 1997). The 19S regulatory complex is composed of two subcomplexes, the lid, which seems to bind and disassemble the ubiquitin-conjugated substrate, and the base, which contains six homologous ATPase subunits (termed Rpt1–6 in yeast) plus two non-ATPases, Rpn 1 and 2 (Voges et al., 1999). These ATPases are members of the AAA family of ATPases (Patel and Latterich, 1998). For a globular protein to be degraded, it must associate with the 19S ATPases and undergo ATP-dependent unfolding followed by translocation into the 20S particle, which requires opening of the gate in the α ring (Kohler et al., 2001). Each of these steps is regulated in some way by the ATPase complex.

The ATPase complexes that regulate protein degradation in eukaryotes, bacteria and archaea are all members of the AAA+ (ATPases Associated with various cellular Activities) ATPase superfamily (for review see Ogura and Tanaka, 2003). The AAA+ family of ATPases are found in all living organisms and in all cell compartments, where they participate in a variety of essential cellular processes such as mitosis, protein folding and translocation, DNA replication and repair, membrane fusion and proteolysis. They are characterized by the presence of one or two conserved ATP-binding domains (200–250 residues), called the AAA motif, consisting of a Walker A and a Walker B motif (Confalonieri and Duguet, 1995). The eukaryotic and archaeal (PAN) proteasomal ATPases belong to a subfamily of AAA+ ATPases (AAA family) that contains an additional motif called the second region of homology (SRH) (Lupas and Martin, 2002). Despite the large variety of cellular processes in which AAA+ ATPases participate, they have some common features. A recurrent structural feature of most AAA+ ATPases is their assembly into oligomeric (generally hexameric) ring-shaped structures with a central pore. In addition, most appear to be involved in protein folding or unfolding, and assembly or disassembly of protein complexes through nucleotide-dependent conformational changes. Thus, recent insights into the functioning of the archaeal PAN complex and the 19S proteasomal regulatory ATPase may illuminate the functioning of these other AAA Family members (and vise versa).

Though bacteria do not contain 20S proteasomes, like those in eukaryotes, they do contain several large compartmentalized protease complexes that associate with AAA ATPase complexes such as HslUV and ClpAP. HslV is a two-ring peptidase complex which shares homology with the beta subunits of the 20S proteasome (Bochtler et al., 2000), and forms a six-membered ring (Rohrwild et al., 1997) rather than the seven-membered ring, which is characteristic of the 20S proteasome. HslU, the ATPase complex, associates with HslV to stimulate protein degradation, and is homologous to PAN. X-ray diffraction studies established that HslU induces conformational changes in the peptidase active site of HslV upon association and increases the pore size of HslV. Thus, HslU increases the peptidase activity of HslV by allosteric activation and probably also by promoting substrate unfolding for peptide entry (Huang and Goldberg, 1997, Sousa et al., 2000, Wang et al., 2001, Yoo et al., 1997). Facilitating peptide entry thus appears to be a common property shared by HslU, PAN, and the 19S ATPases, although HslV does not contain an outer α ring or gating termini like those in the 20S proteasome.

Section snippets

The function of the 26S ATPases

Studying the ATP-dependent processes and the mechanisms of protein breakdown within the 26S proteasome has proven difficult because of its structural complexity, multiple enzymatic activities and ubiquitin requirement. Nevertheless, several important discoveries about these ATPases have been made using genetic tools in yeast. Through systematic mutagenesis of the ATP binding sites in each of the six different ATPases, Finley and co-workers showed that these 19S ATPases (Rpt1–6) perform distinct

The PAN ATPase complex from archaea

The first complete genome sequence in the domain of archaea was from Methanococcus jannaschii. This sequencing revealed a gene (S4) which was highly homologous to the genes encoding the 19S ATPases (Bult et al., 1996). To test if this gene product might regulate the 20S proteasome, the S4 gene was expressed in E. coli, and the 50kDa product, named PAN (proteasome-activating nucleotidase), was purified and characterized by Zwickl et al. (1999). PAN’s sequence contains several hallmarks of the

Structure and association of PAN with the 20S particle

Although the association of an ATPase chaperone-like complex with a proteolytic particle appears to be a common feature of several ATP-dependent systems for intracellular protein degradation (the 26S proteasome and the bacterial ClpAP, ClpXP and HslUV complexes), an association between PAN and the 20S particle was difficult to observe by typical biochemical approaches, even when PAN and the 20S came from the same species. However, when PAN is mixed with archaeal 20S proteasomes and ATP, it

PAN regulates gate opening

Because of the tight interaction between the 20S proteasomes α and β subunits, substrates can enter only through the 20S pore at either end of the particle (Baumeister et al., 1998). The elegant X-ray analysis of M. Groll et al. showed that this channel is gated by the N-termini of the α subunits (Groll et al., 1997). These N-termini in eukaryotic proteasomes can assume either of two ordered structures, an open conformation and a closed one, both of which require the YDR motif for stabilization

The mechanism of the PAN–20S association and gate opening

These studies have established that ATP-binding to PAN is essential for its association with the 20S particle and for triggering gate opening in the 20S (Smith et al., 2005). A very different (non-homologous) type of proteasomal regulator, the 11S, PA28 (REG) complex and its invertebrate homolog, PA26, also bind to the α-ring (independent of ATP) and induce gate opening. However, these complexes stimulate peptide but not protein entry. Their association with the 20S requires their extreme

The energy requirements for protein unfolding and translocation

It has been clear since the early seventies that protein degradation in prokaryotes and eukaryotes requires ATP (Ciechanover, 2005, Goldberg, 2005, Goldberg and St. John, 1976). However, it is still unclear which of the multiple steps in the process of protein degradation requires ATP and how nucleotide binding or hydrolysis enhance these steps. In particular, determining the mechanisms whereby the 19S regulatory particle unfolds substrates and facilitates their entry into the 20S proteolytic

Conclusion

A more complete understanding of the molecular mechanisms involved in protein degradation by the proteasome will be aided by detailed structural information about the ATP and ADP-bound forms of ATPase complex, as well as the delineation of its ATP hydrolysis cycle. Presumably X-ray crystallography will first be achieved for PAN, whose many benefits for study have been summarized here. Already however, study of the PAN:20S complex has allowed us to learn much concerning the multiple steps in

References (78)

  • B.S. Glick

    Can Hsp70 proteins act as force-generating motors?

    Cell

    (1995)
  • A.L. Goldberg

    Nobel committee tags ubiquitin for distinction

    Neuron

    (2005)
  • M. Groll et al.

    Investigations on the maturation and regulation of archaebacterial proteasomes

    J. Mol. Biol.

    (2003)
  • M. Groll et al.

    Substrate access and processing by the 20S proteasome core particle

    Int. J. Biochem. Cell Biol.

    (2003)
  • R. Hough et al.

    Purification of two high molecular weight proteases from rabbit reticulocyte lysate

    J. Biol. Chem.

    (1987)
  • H. Huang et al.

    Proteolytic activity of the ATP-dependent protease HslVU can be uncoupled from ATP hydrolysis

    J. Biol. Chem.

    (1997)
  • A.V. Kajava

    What curves alpha-solenoids? Evidence for an alpha-helical toroid structure of Rpn1 and Rpn2 proteins of the 26 S proteasome

    J. Biol. Chem.

    (2002)
  • J.A. Kenniston et al.

    Linkage between ATP consumption and mechanical unfolding during the protein processing reactions of an AAA+ degradation machine

    Cell

    (2003)
  • A.F. Kisselev et al.

    Range of sizes of peptide products generated during degradation of different proteins by archaeal proteasomes

    J. Biol. Chem.

    (1998)
  • A.F. Kisselev et al.

    Why does threonine, and not serine, function as the active site nucleophile in proteasomes

    J. Biol. Chem.

    (2000)
  • A. Kohler et al.

    The axial channel of the proteasome core particle is gated by the Rpt2 ATPase and controls both substrate entry and product release

    Mol. Cell

    (2001)
  • C. Lee et al.

    ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal

    Mol. Cell

    (2001)
  • A.N. Lupas et al.

    AAA proteins

    Curr. Opin. Struct. Biol.

    (2002)
  • C.P. Ma et al.

    PA28, an activator of the 20 S proteasome, is inactivated by proteolytic modification at its carboxyl terminus

    J. Biol. Chem.

    (1993)
  • K.E. Matlack et al.

    BiP acts as a molecular ratchet during posttranslational transport of prepro-alpha factor across the ER membrane

    Cell

    (1999)
  • A. Matouschek

    Protein unfolding—an important process in vivo?

    Curr. Opin. Struct. Biol.

    (2003)
  • K. Murakami et al.

    Protein degradation is stimulated by ATP in extracts of Escherichia coli

    J. Biol. Chem.

    (1979)
  • Y. Murakami et al.

    Degradation of ornithine decarboxylase by the 26S proteasome

    Biochem. Biophys. Res. Commun.

    (2000)
  • A. Navon et al.

    Proteins are unfolded on the surface of the ATPase ring before transport into the proteasome

    Mol. Cell

    (2001)
  • T. Ogura et al.

    Dissecting various ATP-dependent steps involved in proteasomal degradation

    Mol. Cell

    (2003)
  • R.T. Sauer et al.

    Sculpting the proteome with AAA(+) proteases and disassembly machines

    Cell

    (2004)
  • D.M. Smith et al.

    ATP binding to PAN or the 26S ATPases causes association with the 20S proteasome, gate opening, and translocation of unfolded proteins

    Mol. Cell

    (2005)
  • M.C. Sousa et al.

    Crystal and solution structures of an HslUV protease-chaperone complex

    Cell

    (2000)
  • E. Strickland et al.

    Recognition of misfolding proteins by PA700, the regulatory subcomplex of the 26 S proteasome

    J. Biol. Chem.

    (2000)
  • J. Walz et al.

    26S proteasome structure revealed by three-dimensional electron microscopy

    J. Struct. Biol.

    (1998)
  • J. Wang et al.

    Nucleotide-dependent conformational changes in a protease-associated ATPase HsIU

    Structure

    (2001)
  • L. Waxman et al.

    Demonstration of two distinct high molecular weight proteases in rabbit reticulocytes, one of which degrades ubiquitin conjugates

    J. Biol. Chem.

    (1987)
  • S.J. Yoo et al.

    ATP binding, but not its hydrolysis, is required for assembly and proteolytic activity of the HslVU protease in Escherichia coli

    Biochem. Biophys. Res. Commun.

    (1997)
  • M. Zhang et al.

    Repeat sequence of Epstein–Barr virus-encoded nuclear antigen 1 protein interrupts proteasome substrate processing

    J. Biol. Chem.

    (2004)
  • Cited by (0)

    View full text