Proteasomes and Rpt Subunits

Protein degradation by proteasomes is a major regulatory mechanism in eukarya, archaea, and actinobacteria. Proteasomes can degrade short-lived proteins that control cell division, apoptosis, DNA repair, information processing (transcription and translation), and other cellular processes (Navon and Ciechanover 2009). Proteasomes are also important in maintaining protein quality by destroying improperly synthesized, foreign, and damaged proteins (Schrader et al. 2009).

All proteasomes are composed of a 20S core particle (CP) lined on its interior with proteolytic active sites that are sequestered away from the cytosol (Stadtmueller and Hill 2011) (Fig. 1). CPs are barrel-like and formed from four-stacked heptameric rings of α- and β-type subunits. The α-type subunits form the outermost rings, while the β-type subunits form the inner two rings. The N-terminal tails of α-type subunits can gate the pores on each end of CPs and limit substrate access to the proteolytic active sites formed by β-type subunits.

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CPs associate with various regulators (Stadtmueller and Hill 2011). In eukarya, CPs bind 19S regulatory particles (RPs) to form an energy-dependent protease known as the 26S proteasome (Fig. 2). Substrates of 26S proteasomes include proteins covalently conjugated to chains of ubiquitin (Ub) through a process termed ubiquitylation (Ravid and Hochstrasser 2008). The primary roles of 19S RPs are to recognize, unfold, and translocate substrate proteins to the catalytic central chamber of CPs for protein degradation. In yeast, the 19S RP can be separated into base and lid subcomplexes by deletion of the gene encoding the regulatory particle non-ATPase Rpn10 subunit (Tomko and Hochstrasser 2011). The base subcomplex is composed of three Rpn subunits (Rpn1, Rpn2, and Rpn13) and six proteins termed regulatory particle triple-A subunits (Rpt1-6) that cluster to the AAA family of the AAA+ superfamily. Rpt1-6 form a heterohexameric ring that directly interacts with 20S CPs and are arranged in Rpt1-Rpt2-Rpt6-Rpt3-Rpt4-Rpt5 order in 26S proteasomes (Tomko and Hochstrasser 2011). The lid subcomplex is composed of nine other Rpn subunits and harbors the deubiquitylating enzyme Rpn11 that removes polyubiquitin chains from substrate proteins.

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Archaea and actinobacteria synthesize proteasomes that are relatively simple in subunit complexity, yet these proteases share many basic structural and functional features with eukaryal 26S proteasomes (Bar-Nun and Glickman 2011). Like eukarya, the CPs and proteasomal AAA+ proteins of archaea and actinobacteria can associate together in vitro and catalyze the energy-dependent degradation of folded proteins (Fig. 2). The prokaryotic AAA+ proteins are often hexameric rings formed from a single protein including the archaeal proteasome-associated nucleotidase (PAN) and actinobacterial AAA ATPase forming ring-shaped complexes (ARC) or mycobacterium proteasome ATPase (Mpa). The archaeal PAN is closely related to the eukaryal Rpt subunits, while the actinobacterial ARC/Mpa proteins are divergent members of the AAA family. It has been proposed that the six different types of eukaryal Rpt proteins (Rpt1-6) evolved from a single archaeal PAN ancestor, which over time was duplicated and diversified (Wollenberg and Swaffield 2001).

Rpt and Related AAA+ Proteins in Proteolysis

While the hydrolysis of peptide bonds is exergonic, ATP binding and hydrolysis by AAA+ proteins (or domains) is needed to fuel the regulated degradation of folded proteins by self-compartmentalized proteases such as 26S proteasomes. The AAA+ rings can bind substrate proteins, open the gates of the protease chamber (closed-gates limit substrate access to the proteolytic active sites), unfold substrate proteins, and facilitate the translocation of protein substrates into the proteolytic center of the protease.

Subdomain X-ray crystal and cryo-electron microscopy structures of proteasomal AAA+ proteins are now available and provide valuable insight into how this group of ATPases might function at the atomic level (Bar-Nun and Glickman 2011). Details on the related bacterial AAA+ proteases such as ClpXP and HslUV have also guided proteasomal models (Sauer and Baker 2011). In general, proteasomal ATPases have a central channel that is coaxial to the 20S CP channel (Fig. 3). On the distal face of the proteasomal ATPase ring are six protruding and paired N-terminal coiled-coil (CC) domains that are required for substrate binding. Moving down the ATPase channel, one finds a conserved interdomain region with an oligonucleotide-binding (OB)-fold domain that is important for AAA+ ring formation. The OB-fold domain also appears to serve as a rigid entryway for substrates that traverse the ATPase channel. The archaeal PAN and eukaryal Rpt1-6 proteins have a single OB-fold (as depicted in Fig. 3), while the actinobacterial ARC/Mpa has a double OB-fold that forms two separate ring-like structures within the ATPase channel. The C-terminal AAA+ domain mediates cycles of ATP hydrolysis and harbors a highly conserved aromatic-hydrophobic (Ar-phi) loop within its channel. The Ar-phi loop is common to energy-dependent AAA+ proteases and is thought to grab onto hydrophobic tails of substrates that may extend into the ATPase channel. Once bound, cycles of ATP hydrolysis around the ATPase ring are thought to mediate conformational changes in the Ar-phi loop that result in the tugging and unfolding of the substrate protein. The rigid OB-fold pore is believed to serve as a platform for this unfolding process. Thus, the protein substrate is thought to be unfolded and translocated through the ATPase and into the CP for destruction.

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Many of the proteasomal ATPases (e.g., archaeal PAN and eukaryal Rpt2, 3 and 5) have C-terminal hydrophobic-tyrosine-any residue (HbYX) motifs that bind to pockets between the α-type subunits of CPs and induce CP gate opening. The proteasome-associated regulator Blm10 and α-ring assembly factor Pba1-Pba2 also have penultimate tyrosine (or phenylalanine) residues that appear important for binding within the α/α-intersubunit interface (Stadtmueller and Hill 2011; Kusmierczyk et al. 2011).

Substrate Recognition by Proteasomes

Protein degradation is a highly regulated process. In order to ensure that only the desired proteins are degraded, eukaryotes and prokaryotes have evolved various strategies for identifying proteins for degradation. Specific degradation signals or “degrons” within the protein often initiate the process of proteolysis. Degrons range from the phosphorylation or glycosylation status of a protein to the exposure of destabilizing N- or C-termini (Ravid and Hochstrasser 2008). In eukaryotic cells, the ubiquitylation system often recognizes these degrons and results in the covalent attachment of polyubiquitin (Ub) chains. While all seven lysine residues of Ub can form chains, Ub chains linked through Lys48 commonly target proteins for destruction by 26S proteasomes (Xu et al. 2009). These ubiquitylated proteins are thought to first bind the Rpn10 and Rpn13 subunits of 26S proteasomes by a mechanism stimulated by ATP binding to the Rpt subunits (Peth et al. 2010) (Fig. 4). Next, Rpt-mediated ATP hydrolysis is thought to unfold and expose unstructured portions of the substrate protein for tighter binding to 26S proteasomes. Ultimately, this is thought to ready the substrate for the editing or removal of polyUb chains by deubiquitylases. The Rpt subunits would unfold and translocate the protein while opening the CP gates for protein degradation.

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In actinobacteria and archaea, the covalent attachment of small protein modifiers to substrate proteins appears to be linked to proteasome-mediated proteolysis– much like the process of ubiquitylation in eukaryotic cells. It has recently been shown in Mycobacterium tuberculosis that a small protein termed Pup can covalently modify proteins at lysine residues (Burns and Darwin 2010). While the mechanism of pupylation differs from ubiquitylation and the disordered structure of Pup is not like the highly ordered beta-grasp fold of Ub, Pup can target proteins for proteasome-mediated degradation (Burns and Darwin 2010). Interestingly, the mycobacterial proteasomal AAA+ Mpa (required for cellular resistance to reactive nitrogen intermediates) binds and induces formation of an alpha helix within Pup and is required for degradation of pupylated substrates by proteasomes (Burns and Darwin 2010). The haloarchaeon Haloferax volcanii can also attach small archaeal protein modifiers or SAMPs to substrate proteins (Humbard et al. 2010). However, unlike Pup, the archaeal SAMPs have a predicted beta-grasp fold structure similar to Ub and require the presence of an E1 Ub activating homolog (UbaA) for attachment (Miranda et al. 2011). Other enzyme (E2 Ub conjugating and E3 Ub ligase) homologs for the attachment of SAMPs to protein targets have yet to be identified. Sampylation has not been directly linked to targeting proteins for proteasome-mediated degradation. However, the sampylome accumulates in strains deficient in the synthesis of the proteasomal Rpt-like PAN-A and CP α1 subunits, suggesting sampylation may trigger proteins for degradation by proteasomes. However, green fluorescent protein (GFP) derivatives with hydrophobic C-terminal tails that are not modified by sampylation or other types of posttranslational modification are degraded in vitro by proteasomal CPs in the presence of archaeal PAN and hydrolyzable ATP (Navon and Goldberg 2001). Therefore, by an ATP-dependent mechanism, Rpt-like proteins can bind and unfold non-sampylated protein substrates with exposed hydrophobic regions. Interestingly, in eukaryotic cells, the chaperonin-like activities of the proteasomal Rpt proteins appear to also serve non-proteolytic roles (Kodadek 2010).