A Membrane-Bound Archaeal Lon Protease Displays ATP-Independent Proteolytic Activity towards Unfolded Proteins and ATP-Dependent Activity for Folded Proteins

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Abstract
In contrast to the eucaryal 26S proteasome and the bacterial ATP-dependent proteases, little is known about the energy-dependent proteolysis in members of the third domain,Archaea. We cloned a gene homologous to ATP-dependent Lon protease from a hyperthermophilic archaeon and observed the unique properties of the archaeal Lon. Lon fromThermococcus kodakaraensisKOD1 (LonTk) is a 70-kDa protein with an N-terminal ATPase domain belonging to the AAA+superfamily and a C-terminal protease domain including a putative catalytic triad. Interestingly, a secondary structure prediction suggested the presence of two transmembrane helices within the ATPase domain and Western blot analysis using specific antiserum against the recombinant protein clearly indicated that LonTkwas actually a membrane-bound protein. The recombinant LonTkpossessed thermostable ATPase activity and peptide cleavage activity toward fluorogenic peptides with optimum temperatures of 95 and 70°C, respectively. Unlike the enzyme fromEscherichia coli, we found that LonTkshowed higher peptide cleavage activity in the absence of ATP than it did in the presence of ATP. When three kinds of proteins with different thermostabilities were examined as substrates, it was found that LonTkrequired ATP for degradation of folded proteins, probably due to a chaperone-like function of the ATPase domain, along with ATP hydrolysis. In contrast, LonTkdegraded unfolded proteins in an ATP-independent manner, suggesting a mode of action in LonTkdifferent from that of its bacterial counterpart.