A Proteasome from the Methanogenic Archaeon Methanosarcina thermophila (original) (raw)
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Structural features of archaebacterial and eukaryotic proteasomes
Molecular Biology Reports, 1995
The 26S proteasome is the central protease of the ubiquitin-dependent pathway of protein degradation. The molecule has a molecular mass of approximately 2000 kD and has a highly conserved structure in eukaryotes. The 26S proteasome is formed by a barrel-shaped 20S core complex and two polar 19S complexes. The 20S complex has C2 symmetry and is formed by four seven-membered rings of which the outer rings (c~-type subunits) are rotated by 25.7 ° relative to the inner rings while the inner rings (/3-type subunits) are in register. From a comparison of the activity and regulation of the 26S and 20S particles it can be deduced that the 20S particle contains the protease activity while the 19S complex contains isopeptidase, ATPase and protein unfolding activities. In this article we describe the structures of various proteasome complexes as determined by electron microscopy and discuss structural implications of their subunit sequences.
Journal of bacteriology, 1998
The 20S proteasome from the methanoarchaeon Methanosarcina thermophila was produced in Escherichia coli and characterized. The biochemical properties revealed novel features of the archaeal 20S proteasome. A fully active 20S proteasome could be assembled in vitro with purified native alpha ring structures and beta prosubunits independently produced in Escherichia coli, which demonstrated that accessory proteins are not essential for processing of the beta prosubunits or assembly of the 20S proteasome. A protein complex with a molecular mass intermediate to those of the alpha7 ring and the 20S proteasome was detected, suggesting that the 20S proteasome is assembled from precursor complexes. The heterologously produced M. thermophila 20S proteasome predominately catalyzed cleavage of peptide bonds carboxyl to the acidic residue Glu (postglutamyl activity) and the hydrophobic residues Phe and Tyr (chymotrypsinlike activity) in short chromogenic and fluorogenic peptides. Low-level hydro...
Journal of Bacteriology, 2007
The hyperthermophilic archaeon Pyrococcus furiosus genome encodes three proteasome component proteins: one ␣ protein (PF1571) and two  proteins (1-PF1404 and 2-PF0159), as well as an ATPase (PF0115), referred to as proteasome-activating nucleotidase. Transcriptional analysis of the P. furiosus dynamic heat shock response (shift from 90 to 105°C) showed that the 1 gene was up-regulated over twofold within 5 minutes, suggesting a specific role during thermal stress. Consistent with transcriptional data, two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that incorporation of the 1 protein relative to 2 into the 20S proteasome (core particle [CP]) increased with increasing temperature for both native and recombinant versions. For the recombinant enzyme, the 2/1 ratio varied linearly with temperature from 3.8, when assembled at 80°C, to 0.9 at 105°C. The recombinant ␣؉1؉2 CP assembled at 105°C was more thermostable than either the ␣؉1؉2 version assembled at 90°C or the ␣؉2 version assembled at either 90°C or 105°C, based on melting temperature and the biocatalytic inactivation rate at 115°C. The recombinant CP assembled at 105°C was also found to have different catalytic rates and specificity for peptide hydrolysis, compared to the 90°C assembly (measured at 95°C). Combination of the ␣ and 1 proteins neither yielded a large proteasome complex nor demonstrated any significant activity. These results indicate that the 1 subunit in the P. furiosus 20S proteasome plays a thermostabilizing role and influences biocatalytic properties, suggesting that  subunit composition is a factor in archaeal proteasome function during thermal stress, when polypeptide turnover is essential to cell survival. . microtiter plates with bovine serum albumin (Sigma-Aldrich, St. Louis, MO) as the standard.
Investigations on the Maturation and Regulation of Archaebacterial Proteasomes
Journal of Molecular Biology, 2003
The 20 S proteasome (core particle, CP) is a multifunctional protease complex and composed of four heptameric subunit rings arranged in a hollow, barrel-shaped structure. Here, we report the crystal structure of the CP from Archaeoglobus fulgidus at 2.25 Å resolution. The analysis of the structure of early and late assembly intermediates of this CP gives new insights in the maturation of archaebacterial CPs and indicates similarities to assembly intermediates observed in eukaryotes. We also show a striking difference in mechanism and regulation of substrate access between eukaryotic and archaebacterial 20 S proteasomes. While eukaryotic CPs are auto-inhibited by the N-terminal tails of the outer a-ring by imposing topological closure with a characteristic sequence motif (YDR-motif) and show regulatory gating this segment is disordered in the CP and differently structured in the a 7 -sub-complex of A. fulgidus leaving a pore leading into the particle with a diameter of 13 Å . Mutagenesis and functional studies indicate the absence of regulatory gating in the archaeal 20 S proteasome.
Nature communications, 2015
In eukaryotes, the covalent attachment of ubiquitin chains directs substrates to the proteasome for degradation. Recently, ubiquitin-like modifications have also been described in the archaeal domain of life. It has subsequently been hypothesized that ubiquitin-like proteasomal degradation might also operate in these microbes, since all archaeal species utilize homologues of the eukaryotic proteasome. Here we perform a structural and biochemical analysis of a ubiquitin-like modification pathway in the archaeon Sulfolobus acidocaldarius. We reveal that this modifier is homologous to the eukaryotic ubiquitin-related modifier Urm1, considered to be a close evolutionary relative of the progenitor of all ubiquitin-like proteins. Furthermore we demonstrate that urmylated substrates are recognized and processed by the archaeal proteasome, by virtue of a direct interaction with the modifier. Thus, the regulation of protein stability by Urm1 and the proteasome in archaea is likely representa...
Proteasomes in the archaea: from structure to function
Frontiers in Bioscience, 2000
Introduction 3. Proteasomes and the 20S proteolytic core 3.1. Architecture of the 20S proteasome 3.2. Catalytic mechanism of peptide bond hydrolysis 3.3. Distribution and subunit composition of 20S proteasomes 3.4. Assembly of 20S proteasomes 4. Proteasome-associated AAA + ATPases 4.1. AAA + superfamily 4.2. Rpt (AAA +) subunits of eucaryal 26S proteasomes 4.3. Archaeal proteasome-activating nucleotidase (PAN) 4.4. Eubacterial AAA ATPase-forming ring-shaped complexes (ARC) 4.5. Chaperone activity of AAA + proteins 5. Substrate targeting and signal recognition 5.1. Ubiquitin-dependent 5.2. Ubiquitin-independent 6. Archaeal genomics 6.1. 20S proteasome and PAN operons 6.2. Additional energy-dependent proteases 6.2.1. Rpt/FtsH ATPase 6.2.2. Lon protease 6.2.3. Clp ATPase and HtrA (DegP) 7. Role of proteasomes in stress responses 7.1. Eucaryal 26S proteasome and stress 7.2. Eubacterial HslUV proteases and stress 7.3. Prokaryotic 20S proteasomes and AAA + proteins and stress 8. Perspective 9. Acknowledgments 10. References
An Archaeal Homolog of Proteasome Assembly Factor Functions as a Proteasome Activator
PLoS ONE, 2013
Assembly of the eukaryotic 20S proteasome is an ordered process involving several proteins operating as proteasome assembly factors including PAC1-PAC2 but archaeal 20S proteasome subunits can spontaneously assemble into an active cylindrical architecture. Recent bioinformatic analysis identified archaeal PAC1-PAC2 homologs PbaA and PbaB. However, it remains unclear whether such assembly factor-like proteins play an indispensable role in orchestration of proteasome subunits in archaea. We revealed that PbaB forms a homotetramer and exerts a dual function as an ATP-independent proteasome activator and a molecular chaperone through its tentacle-like C-terminal segments. Our findings provide insights into molecular evolution relationships between proteasome activators and assembly factors.