Abstract
The gene coding for the integral membrane protein bacterioopsin (Bop), that is composed of seven transmembrane helices, was expressed in the halophilic archaeon Haloferax volcanii as a fusion protein with the halobacterial enzyme dihydrofolate reductase and with the cellulose binding domain of Clostridium thermocellum cellulosome. In each case, bacterioopsin was present both in the membrane and in the cytoplasmic fractions. Pulse-chase labeling experiments showed that the fusion protein in the cytoplasmic fraction is the precursor of the membrane-bound species. Bacterioopsin mutants that lack the seventh helix (BopDelta7) were found to accumulate only in the cytoplasmic fraction, whereas bacterioopsin mutants that lack either helices four and five (BopDelta4-5), or helices one and two (BopDelta1-2), were found in the cytoplasmic as well as in the membrane fractions. The seventh helix, when expressed alone, could target in trans the insertion of a separately expressed bacterioopsin mutant protein that has only the first six helices. These results support a model in which bacterioopsin is produced in H. volcanii as a soluble protein and in which its insertion into the membrane occurs post-translationally. According to this model, membrane insertion is directed by the seventh helix.
Highlights
signal recognition particle (SRP) is not essential for the survival of the yeast cell [10]
The gene coding for the integral membrane protein bacterioopsin (Bop), that is composed of seven transmembrane helices, was expressed in the halophilic archaeon Haloferax volcanii as a fusion protein with the halobacterial enzyme dihydrofolate reductase and with the cellulose binding domain of Clostridium thermocellum cellulosome
Rationale of the Experimental Approach—In the past, the elucidation of purple membrane biogenesis in H. salinarum was approached by the characterization of mutants deficient in purple membrane formation
Summary
H. volcanii white mutant (SX) (kindly obtained from Dr Robert Charlebois, University of Ottawa, Ottawa, Canada), H. volcanii methionine/cysteine auxotrophic strain (WR341), H. salinarum S9-Bop constitutively expressing strain, and H. salinarum L33-Bop deficient strain were used in the present work. Rich medium (H) and agar plates were prepared as described previously [22] with one modification (50 mM Tris-HCl, pH 7.2, was used as the buffer). The medium was supplemented with 1 g/ml novobiocin (Nov) (Sigma). Trimethoprim (Sigma) at a final concentration of 2 g/ml and all-trans-retinal (Sigma) at a final concentration of 10 M were added to the medium when required. Escherichia coli strain TG1 was grown in Luria-Bertani (LB) medium. Ampicillin (Sigma) was added to the medium to a final concentration of 100 g/ml
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