Abstract
In contrast to the extensively studied eukaryal and bacterial protein secretion systems, comparatively less is known about how and which proteins cross the archaeal cell membrane. To identify secreted proteins of the hyperthermophilic archaeon Aeropyrum pernix K1 we used a proteomics approach to analyze the extracellular and cell surface protein fractions. The experimentally obtained data comprising 107 proteins were compared with the in silico predicted secretome. Because of the lack of signal peptide and cellular localization prediction tools specific for archaeal species, programs trained on eukaryotic and/or Gram-positive and Gram-negative bacterial signal peptide data sets were used. PSortB Gram-negative and Gram-positive analysis predicted 21 (1.2% of total ORFs) and 24 (1.4% of total ORFs) secreted proteins, respectively, from the entire A. pernix K1 proteome, 12 of which were experimentally identified in this work. Six additional proteins were predicted to follow non-classical secretion mechanisms using SecP algorithms. According to at least one of the two PSortB predictions, 48 proteins identified in the two fractions possess an unknown localization site. In addition, more than half of the proteins do not contain signal peptides recognized by current prediction programs. This suggests that known mechanisms only partly describe archaeal protein secretion. The most striking characteristic of the secretome was the high number of transport-related proteins identified from the ATP-binding cassette (ABC), tripartite ATP-independent periplasmic, ATPase, small conductance mechanosensitive ion channel (MscS), and dicarboxylate amino acid-cation symporter transporter families. In particular, identification of 21 solute-binding receptors of the ABC superfamily of the 24 predicted in silico confirms that ABC-mediated transport represents the most frequent strategy adopted by A. pernix for solute translocation across the cell membrane.
Highlights
In contrast to the extensively studied eukaryal and bacterial protein secretion systems, comparatively less is known about how and which proteins cross the archaeal cell membrane
Membrane Surface Proteins of A. pernix K1—Here we carried out a proteomics analysis to identify proteins present on the cell surface of A. pernix K1
One of the most striking features of the surface fraction was the high number of substrate-binding components of transport-related proteins belonging to the ATP-binding cassette (ABC) and tripartite ATP-independent periplasmic (TRAP) systems (Table I and supplemental Table 1)
Summary
Culture Condition—A. pernix K1 (JCM 9820) was grown at 90 °C in a 10-liter fermenter in marine broth (Difco) (37.4 g/liter) and sodium thiosulfate (10 g/liter) as described [17]. Cells were collected by centrifugation at 2000 ϫ g at 4 °C for 30 min and used to prepare the surface fraction. Extraction of Surface Protein Fraction—Cells were washed in 20 mM Tris-HCl, pH 6.5 and resuspended in the same buffer containing 0.7 mM PMSF. The pellet was washed three times with the same buffer, and the collected supernatant containing the surface membrane proteins was harvested and extensively dialyzed against 20 mM Tris-HCl, pH 8.0 and 0.2% Triton X-100. Samples (30 g) were dissolved in 20 l of 125 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 0.2 M DTT, and 0.02% bromphenol blue; boiled at 100 °C for 5 min; and loaded on a 12.5% polyacrylamide gel (10 ϫ 7 cm) [20]. Mascot Server was set up to search the NCBInr-extracted A. pernix K1 database containing 1700. The FASTA format of the genome-translated proteome of A. pernix K1 consisting of 1700 predicted protein sequences was used as the input
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