Proteomic Analysis of the Secretome of Haloarchaeon <i>Natrinema</i> sp. J7–2

Although <i>in silico</i> predictions have revealed that haloarchaea can be distinguished from other organisms in that the Tat pathway is used more extensively than the Sec pathway for haloarchaeal protein secretion, only a few haloarchaeal-secreted proteins have been experimentally confirmed. Here, the culture supernatant and membrane fraction of the haloarchaeon <i>Natrinema</i> sp. J7–2 grown at 23% salt concentration were subjected to RPLC–ESI–MS/MS analysis. In total, 46 predicted Tat substrates, 14 predicted Sec substrates, and 3 class III signal peptide-bearing proteins were detected. Approximately 65% of the detected Tat substrates contain lipoboxes, emphasizing the role of the Tat pathway in haloarchaeal lipoprotein secretion. Most of the detected Tat substrates are extracellular substrate (solute)-binding proteins and redox proteins. Despite the small number of Sec substrates, two of them, a cell surface glycoprotein and a putative lipoprotein carrier protein, were identified to be high-abundance secreted proteins. While limited proteins were detected in the culture supernatant, most of the secreted proteins were found in the membrane fraction. The anchoring of secreted proteins to the cell surface via a lipobox or a PGF-CTERM seems to be an adaptation strategy of haloarchaea to handle the harsh extracellular environment. Additionally, ∼15% of the integral membrane proteins (IMPs) detected in the membrane fraction possess putative Sec signal peptides or signal anchors, implying that the Sec pathway is important for membrane insertion of IMPs. This is the first report to describe the experimental secretome of haloarchaea and provide new information for better understanding of haloarchaeal protein secretion patterns