Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan Universitygrid.49470.3e, Wuhan, China.
State Key Laboratory of Virology, College of Life Sciences, Wuhan Universitygrid.49470.3e, Wuhan, China.
Appl Environ Microbiol. 2022 Apr 26;88(8):e0024622. doi: 10.1128/aem.00246-22. Epub 2022 Mar 29.
In response to high-salt conditions, haloarchaea export most secretory proteins through the Tat pathway in folded states; however, it is unclear why some haloarchaeal proteins are still routed to the Sec pathway. SptE is an extracellular subtilase of sp. strain J7-2. Here, we found that SptE precursor comprises a Sec signal peptide, an N-terminal propeptide, a catalytic domain, and a long C-terminal extension (CTE) containing seven domains (C1 to C7). SptE is produced extracellularly as a mature form (M180) in strain J7-2 and a proform (ΔS) in the Δ mutant strain, indicating that halolysin SptA mediates the conversion of the secreted proform into M180. The proper folding of ΔS is more efficient in the presence of NaCl than KCl. ΔS requires SptA for cleavage of the N-terminal propeptide and C-terminal C6 and C7 domains to generate M180, accompanied by the appearance of autoprocessing product M120 lacking C5. At lower salinities or elevated temperatures, M180 and M120 could be autoprocessed into M90, which comprises the catalytic and C1 domains and has a higher activity than M180. When produced in Haloferax volcanii, SptE could be secreted as a properly folded proform, but its variant (TSptE) with a Tat signal peptide does not fold properly and suffers from severe proteolysis extracellularly; meanwhile, TSptE is more inclined to aggregate intracellularly than SptE. Systematic domain deletion analysis reveals that the long CTE is an important determinant for secretion of SptE via the Sec rather than Tat pathway to prevent enzyme aggregation before secretion. While Tat-dependent haloarchaeal subtilases (halolysins) have been extensively studied, the information about Sec-dependent subtilases of haloarchaea is limited. Our results demonstrate that proper maturation of Sec-dependent subtilase SptE of sp. strain J7-2 depends on the action of halolysin SptA from the same strain, yielding multiple hetero- and autocatalytic mature forms. Moreover, we found that the different extra- and intracellular salt types (NaCl versus KCl) of haloarchaea and the long CTE are extrinsic and intrinsic factors crucial for routing SptE to the Sec rather than Tat pathway. This study provides new clues about the secretion and adaptation mechanisms of Sec substrates in haloarchaea.
在高盐条件下，盐杆菌通过 Tat 途径将大多数分泌蛋白输出到折叠状态；然而，仍然不清楚为什么一些盐杆菌蛋白仍被路由到 Sec 途径。SptE 是 sp. J7-2 菌株的胞外枯草溶菌素。在这里，我们发现 SptE 前体包含 Sec 信号肽、N 端前肽、催化结构域和含有七个结构域（C1 至 C7）的长 C 端延伸（CTE）。SptE 在 J7-2 菌株中以成熟形式（M180）和前体形式（ΔS）在Δ突变株中产生细胞外，表明溶菌素 SptA 介导分泌前体转化为 M180。与 KCl 相比，NaCl 更有利于ΔS 的正确折叠。ΔS 需要 SptA 来切割 N 端前肽和 C 端 C6 和 C7 结构域，以生成 M180，同时出现缺少 C5 的自加工产物 M120。在低盐度或较高温度下，M180 和 M120 可自动加工成 M90，其包含催化和 C1 结构域，比 M180 具有更高的活性。当在 Haloferax volcanii 中产生时，SptE 可以作为正确折叠的前体分泌，但具有 Tat 信号肽的变体（TSptE）不能正确折叠，并在细胞外受到严重的蛋白水解；同时，TSptE 比 SptE 更倾向于在细胞内聚集。系统的结构域缺失分析表明，长 CTE 是通过 Sec 途径而不是 Tat 途径分泌 SptE 的重要决定因素，以防止酶在分泌前聚集。虽然 Tat 依赖性盐杆菌枯草溶菌素（溶菌素）已被广泛研究，但有关盐杆菌 Sec 依赖性枯草溶菌素的信息有限。我们的结果表明，J7-2 菌株的 Sec 依赖性枯草溶菌素 SptE 的正确成熟取决于来自同一菌株的溶菌素 SptA 的作用，产生多种异源和自催化成熟形式。此外，我们发现不同的胞外和胞内盐类型（NaCl 与 KCl）和长 CTE 是 haloarchaea 将 SptE 路由到 Sec 而不是 Tat 途径的重要的外在和内在因素。本研究为 Sec 底物在 haloarchaea 中的分泌和适应机制提供了新的线索。
