Biochemical and Physical Properties of theMethanococcus jannaschii20S Proteasome and PAN, a Homolog of the ATPase (Rpt) Subunits of the Eucaryal 26S Proteasome

Abstract

The 20S proteasome is a self-compartmentalized protease which degrades unfolded polypeptides and has been purified from eucaryotes, gram-positive actinomycetes, and archaea. Energy-dependent complexes, such as the 19S cap of the eucaryal 26S proteasome, are assumed to be responsible for the recognition and/or unfolding of substrate proteins which are then translocated into the central chamber of the 20S proteasome and hydrolyzed to polypeptide products of 3 to 30 residues. All archaeal genomes which have been sequenced are predicted to encode proteins with up to ϳ50% identity to the six ATPase subunits of the 19S cap. In this study, one of these archaeal homologs which has been named PAN for proteasome-activating nucleotidase was characterized from the hyperthermophile Methanococcus jannaschii. In addition, the M. jannaschii 20S proteasome was purified as a 700-kDa complex by in vitro assembly of the ␣ and ␤ subunits and has an unusually high rate of peptide and unfolded-polypeptide hydrolysis at 100°C. The 550-kDa PAN complex was required for CTP-or ATP-dependent degradation of ␤-casein by archaeal 20S proteasomes. A 500-kDa complex of PAN(⌬1-73), which has a deletion of residues 1 to 73 of the deduced protein and disrupts the predicted N-terminal coiled-coil, also facilitated this energy-dependent proteolysis. However, this deletion increased the types of nucleotides hydrolyzed to include not only ATP and CTP but also ITP, GTP, TTP, and UTP. The temperature optimum for nucleotide ( Energy-dependent proteolysis is not only vital to elimination of defective cellular proteins but is also central to regulation of cell division, metabolism, transcription, and other essential cellular functions (12,22). A small group of related energydependent proteases have been identified from several diverse organisms; these proteases include Lon, FtsH (HflB), ClpAP, ClpXP, HslUV (ClpYQ), and the 26S proteasome (4, 39). Although this group of proteins shares limited primary sequence identity, the proteases have converged to a universal structure in which relatively nonspecific proteolytic active sites are compartmentalized from the cell by narrow openings (37, 67). The current model is that the degradation of biological substrates by this group of proteases requires additional energy-dependent proteins or protein domains for the recognition and/or unfolding of substrate proteins (21,29,65). Some of these energy-dependent components may not only participate in proteolysis but also have independent roles as chaperones (60). Thus, the energy-dependent component of these proteases may provide a proofreading step following the initial binding of protein substrates and enable the cell to distinguish between proteins destined for refolding, disaggregation, or destruction (23).Some energy-dependent proteases are organized in a symmetry mismatch, including ClpAP and ClpXP which are complexes of the hexameric ClpA and ClpX ATPases interfaced with the ClpP protease of two heptameric rings (5). It is postulated that hydrolysis of nucleotides...

Role of the β1 Subunit in the Function and Stability of the 20S Proteasome in the Hyperthermophilic ArchaeonPyrococcus furiosus

“…K m values measured were all between 20 and 70 M, which are similar to values for CPs from Rhodococcus and Mycobacterium species (25). V max values obtained were also consistent with previously reported values for other prokaryotic CPs (25,42) and consistent with the native P. furiosus CP (2). Based on the information in Fig.…”

“…The roles of the two ␣ proteins in H. volcanii appear to be different, with separate proteasome structures being assembled based on different stoichiometric ratios of subunits per structure (21,42). In fact, H. volcanii synthesizes at least two native versions of the CP, ␣1ϩ␤ and ␣1ϩ␣2ϩ␤, which may recognize and degrade different types of substrates (21).…”

“…The P. furiosus 20S proteasome ␤ protein complement differs from several other archaeal proteasomes that have been characterized to date (A. fulgidus [15], T. acidophilum [26], M. thermophila [28], M. jannaschii [9,10,42], and H. volcanii [21]), in that it encodes two distinct ␤ protein homologs (Table 3). Original isolation of the proteasome from H. marismortui showed a single ␣ and ␤ composition(11); however, later genome sequencing has revealed the coding possibility for two ␣ and two ␤ proteins.…”

Unfolding the mechanism of the AAA+ unfoldase VAT by a combined cryo-EM, solution NMR study

“…In this way, proteins that have become damaged or that are no longer required can be removed before their accumulation disrupts cellular function (8). A variety of different AAA+ unfoldases have been characterized, including Rpt1-6 in the 19S proteasome regulatory particle in eukaryotes (9)(10)(11), PAN in archaea (12)(13)(14), Mpa/ARC in Actinobacteria (15), ClpA/X (2) and HslU (3) in bacteria, and VAT in the Thermoplasma acidophilum archaebacterium (16)(17)(18)(19)(20).…”

Recombinant ATPases of the yeast 26S proteasome activate protein degradation by the 20S proteasome

“…The Rpt1Rpt2 complex sedimented in glycerol density gradient at the same fraction of Rpt4 complex. Both Rpt1Rpt2 and Rpt4 complexes are of similar size as PAN, which forms a dimerized ring structure of 650 kDa in size [9,18]. Further purification of the Rpt4 and Rpt1Rpt2 complex was not successful because Rpt proteins are easily unstabilized and aggregated.…”