Post-translational Secretion of Fusion Proteins in the Halophilic Archaea Haloferax volcanii

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 peptideencoding 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.

Posttranslational Protein Modification inArchaea

“…Examples of both postand cotranslational translocation have been found in Archaea. Chimeric signal sequence-bearing reporter proteins are secreted posttranslationally from transformed Haloferax volcanii cells (179). In addition, Haloferax volcanii has been reported to posttranslationally insert a chimeric protein containing the multispanning membrane protein bacterio-opsin (318).…”

Membrane Binding of Ribosomes Occurs at SecYE-based Sites in the Archaea Haloferax volcanii

“…In studies involving Haloferax volcanii cells transformed to express chimeric proteins bearing the signal peptide of a native exported protein fused to different reporters, it was shown that protein synthesis and secretion need not be coupled events. 22 In contrast, kinetic radiolabelling approaches revealed the co-translational insertion of the N-terminal portion of the Halobacterium salinarum membrane protein bacterioopsin. 23 Moreover, earlier studies reporting co-sedimentation of SRP (7 S) RNA and bacterioopsin mRNA with membrane-bound polysomes, together with puromycin-induced release of the SRP RNA from the polysomes, 24 as well as more recent studies addressing SRP in H. volcanii 25,26 and other archaeal species, 27 -31 lend further support for the existence of a co-translational mode of protein translocation in Archaea.…”

In the Archaea Haloferax volcanii, Membrane Protein Biogenesis and Protein Synthesis Rates Are Affected by Decreased Ribosomal Binding to the Translocon

“…In Eukarya, SRP is known to couple protein synthesis to protein translocation at the ER membrane (2), whereas membrane protein insertion involves translocon-bound ribosomes (45). Still, examples of post-translational protein translocation in Archaea have been reported (15). Thus, it is tempting to speculate that archaeal protein translocation involves post-translational secretion and co-translational membrane protein insertion.…”

“…The changed membrane protein profile of the transformed cells, relative to the background strain, could reflect the biosynthesis of components involved in post-translational translocation or other proteins in response to this compensatory action. Indeed, post-translational secretion and membrane-protein insertion have been reported to occur in H. volcanii (15,19).…”

“…In the halophilic archaea Haloferax volcanii, secretion of signal sequence-bearing chimeric reporter proteins was shown to occur post-translationally (15), despite the fact that Archaea apparently fail to encode for a SecA homologue (8,9). In contrast, the membrane insertion of Halobacterium salinarum bacterioopsin (Bop), the apoprotein form of the light-driven multispanning proton pump bacteriorhodopsin, relies on a cotranslational process (16 -18).…”