Mechanism For Maturation And Secretion Of Haloarchaeal Tat Substrate SptA

The three domains of life use a variety of pathways for protein secretion. The Sec pathway is employed by most secreted proteins to pass across the cytoplasmic membrane in an unfolded state, while the Tat pathway serves to translocate folded proteins. Haloarchaea export the majority of their secretory proteins via the Tat pathway. Many haloarchaea secrete proteases which enable the degradation of proteins and peptides in the natural environment. The halophilic protease SptA is secreted by haloarchaeon Natrinema sp. J7-2. Mutation analysis of the signal peptide of SptA has shown that it is a Tat-dependent substrate, and the sequence analysis suggests SptA belongs to the subtilisin-like protease (subtilase), known as halolysin, which is usually produced as precursor protein and could autoprocess into the mature enzyme. Whether halolysins can be activated intracellularly and how haloarchaeal cells cope with the risk of intracellular activation of halolysins to avoid proteolysis of cellular proteins are unknown. In the present study, these issues were addressed.Firstly, the recombinant SptA precursor (JAH) and its derivatives were expressed in Escherichia coli, and the maturation process of SptA was investigated. In vitro analysis reveals that the maturation of SptA proceeds via stepwise autoprocessing of its N-terminal propeptide, which acts as an intramolecular chaperone to facilitate the correct folding of SptA. The active mature form is composed of the subtilisin-like catalytic domain and the CTE domain. The CTE domain is not essential for maturation but accelerates the folding and activation process of SptA precursor.Secondly, Haloferax volcanii was used as the host for expression of SptA and its derivatives to investigate whether SptA precursor could be activated within haloarchaeal cell. When expressed in Hfx volcanii, SptA precursor that folded properly in cytosol was secreted efficiently via the Tat pathway. After secretion, the signal peptide was removed by a signal peptidase, releasing the proform to extracellular milieu. Then the proform was autoprocessed into its active mature form. Although most SptA molecules were secreted into the culture medium, minor recombinant SptA could be activated intracellularly and affected the growth of the haloarchaeon. Futhermore, the retardation of SptA secretion in Hfx. volcanii by mutating its Tat signal peptide led to intracellular accumulation of active mature enzyme and caused cell death of the host. These results demonstrate that SptA precursor can be activated intracellularly and accumulation of active mature enzyme intracellularly is lethal to the haloarchaeon.Thirdly, the expression, secretion and maturation of SptA in Natrinema sp. J7-2 were investigated to unravel the relationship between the Tat-dependent SptA secretion and haloarchaeal growth. In Natrinema sp. J7-2, expression and secretion of SptA occurred at the late-log phase and peaked when the culture entered the stationary phase. This indicates that the secretion and activation of SptA is precisely regulated in its native host Natrinema sp. J7-2 to avoid proteolytic damage of cellular proteins at the early stage of cell growth. Besides, SptA is secreted efficiently through Tat pathway that reduces the possibility of its intracellular activation. It was found that the intracellular accumulation of active mature form occurred during the stationary and death phase, and the amount of intracellular mature SptA increased as the culture entered death phase. In this context, the possibility that intracellular accumulation of mature SptA may be involved in the transition of Natrinema sp. J7-2 culture from stationary to death phase can not be excluded.