We report the complete sequence of an extreme halophile, Halobacterium sp. NRC-1, harboring a dynamic 2,571,010-bp genome containing 91 insertion sequences representing 12 families and organized into a large chromosome and 2 related minichromosomes. The Halobacterium NRC-1 genome codes for 2,630 predicted proteins, 36% of which are unrelated to any previously reported. Analysis of the genome sequence shows the presence of pathways for uptake and utilization of amino acids, active sodiumproton antiporter and potassium uptake systems, sophisticated photosensory and signal transduction pathways, and DNA replication, transcription, and translation systems resembling more complex eukaryotic organisms. Whole proteome comparisons show the definite archaeal nature of this halophile with additional similarities to the Gram-positive Bacillus subtilis and other bacteria. The ease of culturing Halobacterium and the availability of methods for its genetic manipulation in the laboratory, including construction of gene knockouts and replacements, indicate this halophile can serve as an excellent model system among the archaea.
The pathway by which segments of a polytopic membrane protein are inserted into the membrane has not been resolved in vivo. We have developed an in vivo kinetic assay to examine the insertion pathway of the polytopic protein bacterioopsin, the apoprotein of Halobacterium salinarum bacteriorhodopsin. Strains were constructed that express the bacteriorhodopsin mutants I4C:H 6 and T5C:H 6 , which carry a unique Cys in the N-terminal extracellular domain and a polyhistidine tag at the C terminus. Translocation of the N-terminal domain was detected using a membrane-impermeant gel shift reagent to derivatize the Cys residue of nascent radiolabeled molecules. Derivatization was assessed by gel electrophoresis of the fully elongated radiolabeled population. The time required to translocate and fully derivatize the Cys residues of I4C:H 6 and T5C:H 6 is 46 ؎ 9 and 61 ؎ 6 s, respectively. This is significantly shorter than the elongation times of the proteins, which are 114 ؎ 26 and 169 ؎ 16 s, respectively. These results establish that translocation of the bacterioopsin N terminus and insertion of the first transmembrane segment occur co-translationally and confirm the use of the assay to monitor the kinetics of polytopic membrane protein insertion in vivo.An essential step in the biogenesis of polytopic or multispanning membrane proteins is the insertion of polypeptide segments into the membrane. To elucidate the insertion mechanism in vivo, the cellular factors that participate in this process must be characterized, and the pathway or sequence with which the polypeptide segments are inserted must be determined.Cellular factors have been identified that mediate protein insertion into the eukaryotic endoplasmic reticulum (1, 2) and bacterial cytoplasmic membranes (3-5). These include the signal recognition particle (SRP), 1 a cytosolic ribonucleoprotein complex, and the secretory translocase, a membrane protein complex. SRP directs ribosome-bound nascent polypeptides to the secretory translocase, which in eukaryotes forms an aqueous channel into which the polypeptides are inserted (6, 7). Sequence conservation of SRP and secretory translocase subunits (8, 9) suggests that aspects of the insertion mechanism are universal.Less is known about the insertion pathway of polytopic membrane proteins. In eukaryotes, a co-translational, sequential insertion pathway (10, 11) is favored (12). In support of this model, the insertion of transmembrane segments of polytopic membrane proteins has been shown to be mechanistically coupled to translation (13-15) and to occur sequentially (15-17). However, other evidence contradicts this model. At least one eukaryotic polytopic membrane protein appears to insert posttranslationally (18), and several others exhibit multiple topologies that may reflect a nonsequential insertion pathway (19 -21). In bacteria, it is not known whether insertion is cotranslational and sequential or more closely resembles protein secretion, where translocation is independent from elongation (22, 23). There is evid...
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