Isolation of fusion proteins containing SecY and SecE, components of the protein translocation complex from the halophilic archaeon Haloferax volcanii

Irihimovitch, Vered; Ring, Gabriela; Elkayam, Tsiona; Konrad, Zvia; Eichler, Jerry

doi:10.1007/s00792-002-0297-0

Cited by 23 publications

(28 citation statements)

References 41 publications

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“…Recent examples are the usage of the BgaH reporter enzyme for the in vivo analysis of constitutive and regulated promoters, and the development of methods for gene inactivation (Gregor & Pfeifer, 2005;Allers et al, 2004). A second possibility is to find a mesohalic protein that is salt-tolerant, and a recent example is the application of a bacterial cellulose-binding domain for affinity purification of fusion proteins and protein complexes from haloarchaea (Irihimovitch et al, 2003). A third possibility is to modify a mesohalic protein until it can withstand high salt conditions; a recent example is the application of a modified GFP for the in vivo analysis of proteasome function (Reuter & Maupin-Furlow, 2004).…”

Section: 'Halophilic Adaptation' Of Methodologiesmentioning

confidence: 99%

From genomes to function: haloarchaea as model organisms

Soppa

2006

Microbiology

124

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Haloarchaea are adapted to high-salt environments and accumulate equally high salt concentrations in the cytoplasm. The genomes of representatives of six haloarchaeal genera have been fully or partially sequenced, allowing the analysis of haloarchaeal properties in silico. Transcriptome and proteome analyses have been established for Halobacterium salinarum and Haloferax volcanii. Genetic systems are available including methods that allow the fast in-frame deletion or modification of chromosomal genes. The high-efficiency transformation system of Hf. volcanii allows the isolation of genes essential for a biological process by complementation of loss-of-function mutants. For the analysis of haloarchaeal biology many molecular genetic, biochemical, structural and cell biological methods have been adapted to application at high salt concentrations. Recently it has become clear that several different mechanisms allow the adaptation of proteins to the high salt concentration of the cytoplasm. Taken together, the wealth of techniques available make haloarchaea excellent archaeal model species.

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Section: 'Halophilic Adaptation' Of Methodologiesmentioning

confidence: 99%

From genomes to function: haloarchaea as model organisms

Soppa

2006

Microbiology

124

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“…Earlier studies have shown these chimeras to be expressed stably and localized exclusively to the membranes of the transformed haloarchaeal cells. 43 In such IMVs, the presence of the CBD moiety, fused to the cytoplasmically facing N terminus of H. volcanii SecE and SecY, would be expected to present a steric hindrance to any ribosomal binding at sites incorporating the CBD-SecE or CBD-SecY chimeras.…”

Section: H Volcanii Ribosomes Bind To Protein Sites In the Membranementioning

confidence: 99%

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

Ring

Eichler

2004

Journal of Molecular Biology

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“…This suggests that SP-CBD is first completely synthesized inside the cells and only then secreted across the plasma membrane into the growth medium. teolysis of heterologously expressed fusion proteins in H. volcanii (22,29,30). Subcellular fractionation of the SP-CBDexpressing cells revealed that the preprotein and its derived breakdown products were restricted to the cytoplasmic fraction (data not shown).…”

Section: Methodsmentioning

confidence: 96%

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

Irihimovitch

Eichler

2003

Journal of Biological Chemistry

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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.

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Isolation of fusion proteins containing SecY and SecE, components of the protein translocation complex from the halophilic archaeon Haloferax volcanii

Cited by 23 publications

References 41 publications

From genomes to function: haloarchaea as model organisms

From genomes to function: haloarchaea as model organisms

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

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

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