Biochemical and physical properties of the Methanococcus jannaschii 20S proteasome and PAN, a homolog of the ATPase (Rpt) subunits of the eucaryal 26S proteasome

The 20S proteasome is a self-compartmentalized protease which degrades unfo lded polypeptides and has been purified from eucaryotes, gram-positive acti nomycetes, and archaea. Energy-dependent complexes, such as the 19S cap of the eucaryal 26S proteasome, are assumed to be responsible for the recognit ion and/or unfolding of substrate proteins which are then translocated into the central chamber of the 20S proteasome and hydrolyzed to polypeptide pr oducts of 3 to 30 residues, All archaeal genomes which have been sequenced are predicted to encode proteins with up to similar to 50% identity to the six ATPase subunits of the 19S cap. In this study, one of these archaeal ho mologs which has been named PAN for proteasome-activating nucleotidase was characterized from the hyperthermophile Methanococcus jannaschii. In additi on, the M. jannaschii 20S proteasome was purified as a 700-kDa complex by i n vitro assembly of the alpha and beta subunits and has an unusually high r ate of peptide and unfolded-polypeptide hydrolysis at 100 degrees C. The 55 0-kDa PAN complex was required for CTP- or ATP-dependent degradation of bet a-casein by archaeal 20S proteasomes. A 500-kDa complex of PAN(Delta 1-73), which has a deletion of residues 1 to 73 of the deduced protein and disrup ts the predicted N-terminal coiled-coil, also facilitated this energy-depen dent 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 (ATP) hydrolysis was reduced from 80 degrees C for the full-length protein to 65 degrees C for PAN(Delta 1-73 ). Both PAN protein complexes were stable in the absence of ATP and were in hibited by N-ethylmaleimide and p-chloromercuriphenyl-sulfonic acid. Kineti c analysis reveals that the PAN protein has a relatively high V-max for ATP and CTP hydrolysis of 3.5 and 5.8 mu mol of P-i per min per mg of protein as well as a relatively low affinity for CTP and ATP with K-m values of 307 and 497 mu M compared to other proteins of the AAA family. Based on electr on micrographs, PAN and PAN(Delta 1-73) apparently associate with the ends of the 20S proteasome cylinder. These results suggest that the M. jannaschi i as well as related archaeal 20S proteasomes require a nucleotidase comple x such as PAN to mediate the energy-dependent hydrolysis of folded-substrat e proteins and that the N-terminal 73 amino acid residues of PAN are not ab solutely required for this reaction.