title: Autocatalytic maturation of the Tat-dependent halophilic subtilase Nep produced by the archaeon Natrialba magadii.
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.
authors.affiliation: Instituto de Investigaciones Biológicas, Facultad de Ciencias Exactas y Naturales Universidad Nacional de Mar del Plata-CONICET, Mar del Plata, Argentina.
authors.forename: Diego M
authors.initials: DM
authors.lastname: Ruiz
authors.forename: Roberto A
authors.initials: RA
authors.lastname: Paggi
authors.forename: Maria I
authors.initials: MI
authors.lastname: Giménez
authors.forename: Rosana E
authors.initials: RE
authors.lastname: De Castro
mesh_terms: D001105:Archaea / Q000235:genetics / Q000378:metabolism*; D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism*; D019848:Gene Expression Regulation, Archaeal / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D021381:Protein Transport; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*
chemical_list: D019843:Archaeal Proteins; D012697:Serine Endopeptidases