Regulatory particle triple-A (Rpt) subunits are associated with the ubiquitin-proteasome system, a central mechanism of protein breakdown with a rich historical background. Aaron Ciechanover, Avram Hershko, and Irwin Rose were awarded the 2004 Nobel Prize in Chemistry for their “discovery of ubiquitin-mediated protein degradation” by proteasomes and have outlined some of this history in reviews (e.g., Ciechanover 2010). The term “proteasome” was first coined by Arrigo et al. (1988) for a “large alkaline multifunctional protease” known to mediate energy-dependent intracellular protein breakdown, once it was clear that this protease was related to the ring-shaped “prosomes” of unknown function commonly found in eukaryotic cells. The Rpt nomenclature was later proposed by Finley et al. (1998) to designate the subunits of the yeast 26S proteasome that cluster to the ATPases associated with various cellular activities (AAA) family of the AAA+ superfamily (Wollenberg and Swaffield 2001). AAA+ proteins are required for the degradation of folded proteins by self-compartmentalized proteases such as proteasomes.
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).
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.
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
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).
Proteins of the AAA+ superfamily that include eukaryal Rpt, archaeal PAN, and actinobacterial Mpa/ARC are structurally related and important in protein degradation. These proteasomal ATPases can unfold proteins, open CP gates, and translocate proteins for ultimate destruction within the self-compartmentalized 20S CP structure that protects cells from uncontrolled proteolysis. In eukarya, Rpt1-6 associate with non-ATPase subunits in complexes such as the 19S RP to form 26S proteasomes but also appear to serve non-proteolytic roles. Whether the archaeal PAN and actinobacterial Mpa/ARC are similar to eukaryal Rpt in their association with proteasomal CPs and other non-ATPase factors in vivo remains an exciting area of research.
This work was funded in part by grants from the National Institutes of Health (GM57498) and the Department of Energy Office of Basic Energy Sciences (DE-FG02-05ER15650).
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