Abstract
Although protein secretion occurs post-translationally in bacteria and is mainly a cotranslational event in Eukarya, the relationship between the translation and translocation of secreted proteins in Archaea is not known. To address this question, the signal peptide-encoding region of the surface layer glycoprotein gene from the Haloarchaea Haloferax volcanii was fused either to the cellulose-binding domain of the Clostridium thermocellum cellulosome or to the cytoplasmic enzyme dihydrofolate reductase from H. volcanii. Signal peptide-cleaved mature versions of both the cellulose-binding domain and dihydrofolate reductase could be detected in the growth medium of transformed H. volcanii cells. Immunoblot analysis revealed, however, the presence of full-length signal peptide-bearing forms of both proteins inside the cytoplasm of the transformed cells. Proteinase accessibility assays confirmed that the presence of cell-associated signal peptide-bearing proteins was not due to medium contamination. Moreover, the pulse-radiolabeled signal peptide cellulose-binding domain chimera could be chased from the cytoplasm into the growth medium even following treatment with anisomycin, an antibiotic inhibitor of haloarchaeal protein translation. Thus, these results provide evidence that, in Archaea, at least some secreted proteins are first synthesized inside the cell and only then translocated across the plasma membrane into the medium.
Highlights
signal peptide (SP)-bearing Fusion Proteins Are Detected Inside Transformed H. volcanii Cells—To assess the relation between protein translation and translocation in Archaea, H. volcanii WR341 cells were transformed with plasmid pWL-SP-cellulose-binding domain (CBD), encoding a chimeric protein composed of the SP of the H. volcanii surface layer glycoprotein (SLG) fused to the cbd gene, encoding the C. thermocellum cellulosome CBD (Fig. 1B)
H. volcanii cells were transformed to express chimeric preproteins formed from the SP of the H. volcanii SLG fused either to the CBD of the C. thermocellum cellulosome, allowing for salt-insensitive cellulose-based purification of the preprotein and its secreted product, or to H. volcanii dihydrofolate reductase-1 (DHFR-1) to monitor the secretion of a haloarchaeal cytoplasmic protein designed for export
Examination of the protein content of the isolated cells revealed the presence of the full-length preproteins, suggesting that secretion of both the foreign CBD and native DHFR-1 had occurred post-translationally
Summary
The process of protein translocation is well described in bacteria and Eukarya (9 –12), little is known about how proteins cross the plasma membrane of Archaea, the third and most recently described domain of life. Archaea encode components of the translocation complexes found in the bacterial and eukaryal systems, and exported archaeal proteins often contain signal peptides similar to those found in the other two domains [13, 14]. Many Archaea encode homologs of SecDF [17], proteins that, in bacteria, serve to modulate the membrane association of SecA, the translocation complex component responsible for coupling ATP energy to the post-translational movement of preproteins across the plasma membrane [18, 19]. Plasmid encoding H. volcanii SLG Ampr,Novr shuttle vector containing both E. coli and H. volcanii replication origins pWL-Nov vector containing the PrR16 promotor fused to the cbd gene encoding the
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