ABSTRACT
Translation initiation in Archaea combines aspects of the parallel process in Eukarya and Bacteria alongside traits unique to this domain. To better understand translation initiation in Archaea, an in vitro translation system from the haloarchaeon Haloferax volcanii has been developed. The ability to translate individual mRNAs both under the conditions used in previously developed poly(U)-dependent poly(Phe) synthesis systems as well as under physiological conditions was shown. Using the H. volcanii system, mRNAs proceeded by either 'strong' or 'weak' Shine-Dalgarno (SD) motifs, or completely lacking leader sequences were effectively translated. The in vitro haloarchaeal system also successfully translated mRNA from Bacteria, again either presenting a SD initiation motif or completely lacking a leader sequence. Thus, the ability to translate individual mRNAs in vitro offers a system to address translation initiation as well as other aspects of protein biogenesis in Archaea.
Subject(s)
Archaea/genetics , Haloferax volcanii/genetics , Protein Biosynthesis/genetics , Amino Acid Motifs/genetics , Archaea/metabolism , Archaeal Proteins/genetics , Archaeal Proteins/metabolism , Electrophoresis, Polyacrylamide Gel , Haloferax volcanii/metabolism , Immunoblotting , Peptide Chain Initiation, Translational , RNA, Archaeal/genetics , RNA, Archaeal/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Ribosomes/metabolism , Transcription, GeneticABSTRACT
In the haloarchaea Haloferax volcanii, ribosomes are found in the cytoplasm and membrane-bound at similar levels. Transformation of H. volcanii to express chimeras of the translocon components SecY and SecE fused to a cellulose-binding domain substantially decreased ribosomal membrane binding, relative to non-transformed cells, likely due to steric hindrance by the cellulose-binding domain. Treatment of cells with the polypeptide synthesis terminator puromycin, with or without low salt washes previously shown to prevent in vitro ribosomal membrane binding in halophilic archaea, did not lead to release of translocon-bound ribosomes, indicating that ribosome release is not directly related to the translation status of a given ribosome. Release was, however, achieved during cell starvation or stationary growth, pointing at a regulated manner of ribosomal release in H. volcanii. Decreased ribosomal binding selectively affected membrane protein levels, suggesting that membrane insertion occurs co-translationally in Archaea. In the presence of chimera-incorporating sterically hindered translocons, the reduced ability of ribosomes to bind in the transformed cells modulated protein synthesis rates over time, suggesting that these cells manage to compensate for the reduction in ribosome binding. Possible strategies for this compensation, such as a shift to a post-translational mode of membrane protein insertion or maintained ribosomal membrane-binding, are discussed.
Subject(s)
Archaeal Proteins/physiology , Haloferax volcanii/metabolism , Proteins/metabolism , Ribosomes/metabolism , Archaea/metabolism , Archaeal Proteins/chemistry , Cell Membrane/metabolism , Cellulose/chemistry , Cytoplasm/metabolism , Densitometry , Electrophoresis, Polyacrylamide Gel , Endoplasmic Reticulum/metabolism , Kinetics , Membrane Proteins/metabolism , Protein Binding , Protein Biosynthesis , Protein Structure, Tertiary , Protein Transport , Puromycin/pharmacology , RNA/metabolism , RNA, Ribosomal/chemistry , Ribosomes/chemistry , Salts/pharmacology , Subcellular Fractions , Time FactorsABSTRACT
In all three domains of life, extracytoplasmic proteins must overcome the hurdle presented by hydrophobic, lipid-based membranes. While numerous aspects of the protein translocation process have been well studied in bacteria and eukarya, little is known about how proteins cross the membranes of archaea. Analysis to date suggests that archael protein translocation is a mosaic of bacterial, eukaryal, and archaeal features, as indeed is much of archaeal biology. Archaea encode homologues of selected elements of the bacterial and eukaryal translocation machines, yet lack other important components of these two systems. Other aspects of the archaeal translocation process appear specific to this domain, possibly related to the extreme environmental conditions in which archsea thrive. In the following, current understanding of archaeal protein translocation is reviewed, as is recent progress in reconstitution of the archaeal translocation process in vitro.
Subject(s)
Archaea/physiology , Archaeal Proteins/metabolism , Cell Membrane/physiology , Membrane Proteins/metabolism , Protein Sorting Signals/physiology , Protein Transport/physiology , Biological Transport, ActiveABSTRACT
Across evolution, the signal recognition particle pathway targets extra-cytoplasmic proteins to membranous translocation sites. Whereas the pathway has been extensively studied in Eukarya and Bacteria, little is known of this system in Archaea. In the following, membrane association of FtsY, the prokaryal signal recognition particle receptor, and SRP54, a central component of the signal recognition particle, was addressed in the halophilic archaea Haloferax volcanii. Purified H. volcanii FtsY, the FtsY C-terminal GTP-binding domain (NG domain) or SRP54, were combined separately or in different combinations with H. volcanii inverted membrane vesicles and examined by gradient floatation to differentiate between soluble and membrane-bound protein. Such studies revealed that both FtsY and the FtsY NG domain bound to H. volcanii vesicles in a manner unaffected by proteolytic pretreatment of the membranes, implying that in Archaea, FtsY association is mediated through the membrane lipids. Indeed, membrane association of FtsY was also detected in intact H. volcanii cells. The contribution of the NG domain to FtsY binding in halophilic archaea may be considerable, given the low number of basic charges found at the start of the N-terminal acidic domain of haloarchaeal FtsY proteins (the region of the protein thought to mediate FtsY-membrane association in Bacteria). Moreover, FtsY, but not the NG domain, was shown to mediate membrane association of H. volcanii SRP54, a protein that did not otherwise interact with the membrane.
Subject(s)
Cell Membrane/metabolism , Haloferax volcanii/metabolism , Signal Recognition Particle/metabolism , Amino Acid Sequence , Archaea/metabolism , Arginine/chemistry , Bacterial Proteins/chemistry , Cytoplasm/metabolism , Endopeptidase K/chemistry , Escherichia coli/metabolism , Immunoblotting , Lysine/chemistry , Molecular Sequence Data , Plasmids/metabolism , Protein Binding , Protein Structure, Tertiary , Receptors, Cytoplasmic and Nuclear/chemistry , Subcellular Fractions/metabolism , Time FactorsABSTRACT
Whereas ribosomes bind to membranes at eukaryal Sec61alphabetagamma and bacterial SecYEG sites, ribosomal membrane binding has yet to be studied in Archaea. Accordingly, functional ribosomes and inverted membrane vesicles were prepared from the halophilic archaea Haloferax volcanii. The ability of the ribosomes to bind to the membranes was determined using a flotation approach. Proteolytic pretreatment of the vesicles, as well as quantitative analyses, revealed the existence of a proteinaceous ribosome receptor, with the affinity of binding being comparable to that found in Eukarya and Bacteria. Inverted membrane vesicles prepared from cells expressing chimeras of SecE or SecY fused to a cytoplasmically oriented cellulose-binding domain displayed reduced ribosome binding due to steric hindrance. Pretreatment with cellulose drastically reduced ribosome binding to chimera-containing but not wild-type vesicles. Thus, as in Eukarya and Bacteria, ribosome binding in Archaea occurs at Sec-based sites. However, unlike the situation in the other domains of Life, ribosome binding in haloarchaea requires molar concentrations of salt. Structural information on ribosome-Sec complexes may provide insight into this high salt-dependent binding.
Subject(s)
Carrier Proteins/metabolism , Cell Membrane/metabolism , Haloferax volcanii/chemistry , Haloferax volcanii/genetics , Membrane Proteins/metabolism , Ribosomes/metabolism , Archaea/chemistry , Archaea/genetics , Archaea/ultrastructure , Binding Sites , Cellulose/metabolism , Cellulose/pharmacology , Haloferax volcanii/ultrastructure , Liposomes , Receptors, Cytoplasmic and Nuclear/metabolism , Sodium Chloride/pharmacologyABSTRACT
By exploiting the salt-insensitive interaction of the cellulose-binding domain (CBD) of the Clostridium thermocellum cellulosome with cellulose, purification of CBD-fused versions of SecY and SecE, components of the translocation apparatus of the halophilic archaeon Haloferax volcanii, was undertaken. Following transformation of Haloferax volcanii cells with CBD-SecY- or -SecE-encoding plasmids, cellulose-based purification led to the capture of stably expressed, membrane-bound 68 and 25 kDa proteins, respectively. Both fusion proteins were recognized by antibodies raised against the CBD. Thus, CBD-cellulose interactions can be employed as a salt-insensitive affinity purification system for the capture of complexes containing the Haloferax volcanii translocation apparatus components SecY and SecE.