Secretion of Tat‐dependent halolysin SptA capable of autocatalytic activation and its relation to haloarchaeal growth

Abstract

SummaryHalolysins are Tat-dependent extracellular subtilases of haloarchaea. Whether halolysins can be activated before transport across the cytoplasmic membrane in a folded state and how haloarchaea minimize the risk of intracellular activation of halolysins and proteolysis of cellular proteins are unknown. Here, we report that both the precursor and proform of halolysin SptA from Natrinema sp. J7-2 mature autocatalytically, and the SptA maturation proceeds less efficiently in the presence of KCl than NaCl. When produced in Haloferax volcanii, most SptA molecules are secreted into the culture medium, but a small number of molecules can be activated intracellularly, affecting the cell's growth. Furthermore, retardation of SptA secretion in Hfx. volcanii via mutation of the Tat signal peptide leads to intracellular accumulation of the active enzyme and subsequent cell death. Although the Sec signal peptide can mediate SptA secretion in Hfx. volcanii, the secreted protein undergoes proteolysis. In Natrinema sp. J7-2, SptA is secreted primarily during stationary phase, and the intracellular accumulation of mature enzyme occurs during the stationary and death phases. The growth phase-dependent synthesis of SptA, highly efficient secretion system, and high intracellular KCl concentration, contribute to the suppression of premature activation of this enzyme in Natrinema sp. J7-2.

Alternative Translation Initiation of a Haloarchaeal Serine Protease Transcript Containing Two In-Frame Start Codons

“…Subsequently, the cell extract was separated from cell debris by centrifugation at 13,400 ϫ g for 10 min at 4°C and used for determination of ␤-galactosidase activity according to the method by Holmes et al (45). The culture supernatants of transformants harboring recombinant plasmids carrying the sptA gene were assayed for azocaseinolytic activity as described previously (28).…”

“…The proteolytic activity of mid-log-phase culture supernatants was assayed using N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (suc-AAPFpNA) (Sigma, St. Louis, MO) as the substrate, as described previously (28). Briefly, enzymatic hydrolysis of suc-AAPF-pNA was carried out at 40°C in buffer C (50 mM Tris-HCl, 3 M NaCl, 10 mM CaCl 2 , pH 8.0) containing the substrate (0.2 mM).…”

“…The signal peptide mediates the translocation of SptA across the cytoplasmic membrane via the Tat pathway, which is used for secretion of folded proteins (30) and then is cleaved by a signal peptidase. The N-terminal propeptide, which functions as an intramolecular chaperone and inhibitor of the mature form, is autocatalytically processed to yield the active mature enzyme composed of the catalytic domain and the CTE (26,28). Notably, it was observed that three in-frame ATGs are present at the 5= end of the sptA gene (25).…”

“…DQ137266). The enzymatic properties, maturation, and secretion mechanisms of SptA and SptC have been characterized (25)(26)(27)(28). As the major extracellular protease of Natrinema sp.…”

“…As the major extracellular protease of Natrinema sp. strain J7, SptA is secreted primarily during stationary phase (28,29). The SptA precursor is composed of a Tat signal peptide, an N-terminal propeptide, a subtilisin-like catalytic domain, and a C-terminal extension (CTE).…”