Abstract
Halolysins are subtilisin-like extracellular proteases produced by haloarchaea that possess unique protein domains and are salt dependent for structural integrity and functionality. In contrast to bacterial subtilases, the maturation mechanism of halolysins has not been addressed. The halolysin Nep is secreted by the alkaliphilic haloarchaeon Natrialba magadii, and the recombinant active enzyme has been synthesized in Haloferax volcanii. Nep contains an N-terminal signal peptide with the typical Tat consensus motif (GRRSVL), an N-terminal propeptide, the protease domain, and a C-terminal domain. In this study, we used Nep as a model protease to examine the secretion and maturation of halolysins by using genetic and biochemical approaches. Mutant variants of Nep were constructed by site-directed mutagenesis and expressed in H. volcanii, which were then analyzed by protease activity and Western blotting. The Tat dependence of Nep secretion was demonstrated in Nep RR/KK variants containing double lysine (KK) in place of the twin arginines (RR), in which Nep remained cell associated and the extracellular activity was undetectable. High-molecular-mass Nep polypeptides without protease activity were detected as cell associated and extracellularly in the Nep S/A variant, in which the catalytic serine 352 had been changed by alanine, indicating that Nep protease activity was needed for precursor processing and activation. Nep NSN 1-2 containing a modification in two potential cleavage sites for signal peptidase I (ASA) was not efficiently processed and activated. This study examined for the first time the secretion and maturation of a Tat-dependent halophilic subtilase.
Highlights
Halolysins are subtilisin-like extracellular proteases produced by haloarchaea that possess unique protein domains and are salt dependent for structural integrity and functionality
In this study we examined the secretion and maturation of halolysins in the model organism H. volcanii, based on protease Nep produced by the haloalkaliphilic archaeon N. magadii
Shi et al had suggested that the protease SptA produced by the haloarchaeon Natrinema sp. strain J7 was likely transported to the extracellular medium through the Tat pathway [28]
Summary
Halolysins are subtilisin-like extracellular proteases produced by haloarchaea that possess unique protein domains and are salt dependent for structural integrity and functionality. Less is known regarding how extracellular proteases are transported through the archaeal membrane and processed into fully active enzymes In this context, the maturation mechanism of the Sec-dependent subtilisin-like proteases secreted by the hyperthermophilic archaeon Thermococcus kodaraensis have been extensively characterized [8, 33]. It was proposed that the extensive use of the Tat secretory pathway in haloarchaea may ensure that secreted proteins are partially or fully folded before translocation, reflecting an evolutionary adaptation to the high salt concentrations that predominate in the environments where these organisms inhabit, as well as inside their cytoplasm [23]. In the extreme halophilic bacteria Salinibacter ruber secreted proteins are predicted to rely on the Sec pathway [5], suggesting that Tat preference is a distinct adaptation of haloarchaea
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