(Received for publication, April 21, 1995; and in revised form, July 31, 1995)
From the
Bacterio-opsin is made as a precursor in Halobacterium halobium, which has 13 additional residues at the amino terminus. The codons for these residues have been proposed to form a hairpin structure in the mRNA and play a role in ribosome binding; the leader peptide sequence also has been proposed to have a role in membrane insertion of bacteriorhodopsin (BR). We have made mutations in the bop gene region coding for the leader sequence and expressed the mutant genes in an H. halobium mutant lacking wild-type BR. The leader sequence coding region was found to be important for the stability of the mRNA and for its efficient translation. Single base substitutions in this region that did not affect the amino acid sequence caused significant reductions in protein expression. Deletion of the leader region resulted in unstable mRNA and almost no BR production. Introduction of a new ribosome-binding sequence within the coding region of the mature protein restored mRNA stability and some protein expression. Protein made without the leader peptide was properly assembled in the membrane.
Bacteriorhodopsin (BR) ()is a light-driven proton
pump in the membrane of the archaeon Halobacterium halobium (also known as Halobacterium salinarium). Bacterio-opsin
(BO), the apoprotein without the retinal chromophore, is made as a
precursor that has 13 additional residues at the amino
terminus(1) . These residues, as well as one at the carboxyl
terminus, are removed after membrane assembly(1, 2) .
The leader sequence does not have the positive amino-terminal and
hydrophobic domain characteristic of both prokaryotic and eukaryotic
signal sequences and is also too short to span the bilayer.
Nevertheless, it has been proposed to have a role in the insertion of
BR into the halobacterial membrane(3, 4) . Mature BR
lacking this sequence inserts spontaneously into phospholipid bilayers in
vitro(5, 6, 7, 8, 9) ,
and BO synthesized in vitro in a wheat germ system is
integrated into dog pancreas microsomes equally well both with and
without the leader sequence(10) . These processes may, however,
be different from what occurs in the H. halobium cell. The
mRNA transcribed from the bop gene coding for BO, as well as
mRNAs for some other H. halobium proteins, starts very close
to the initiation codon and has been proposed to form a stem and loop
secondary structure containing a sequence complementary to the H.
halobium 16S rRNA (Fig. 1) that could act as a
ribosome-binding site within the coding region for the
protein(1, 11, 12) . Our experiments have
confirmed this role for the 5` end of the bop mRNA that codes
for the leader sequence, but we have found no requirement for the
leader sequence itself in membrane insertion or assembly of BR.
Figure 1: Secondary structures and ribosome-binding sites that have been proposed at the 5` end of halobacterial mRNAs. bop, bacterio-opsin(1) ; brp, bacterio-opsin related protein(12) ; hop, halo-opsin(11) .
Figure 2: A, DNA and encoded protein sequences for the wild-type bop gene and the leader sequence deletion mutant genes in pXU10 and pXU10A. Bold characters show the sequence of the mature protein. B, proposed secondary structure for the 5` end of the mRNA in mutant XU10A.
Figure 5:
Proposed structure at the 5` end of the bop mRNA. The bases mutated from G A in strains XU13,
XU14, and XU15 are numbered accordingly and shaded.
Whereas pWL102 is stably maintained in H. halobium, the
presence of a bop gene insert in each case caused the plasmid
to integrate into the chromosome at the site of the endogenous bop gene. DNA from the transformants was analyzed by digestion with SmaI, Southern blotting, and probing with a nick-translated bop gene fragment. SmaI sites are located 450 base
pairs upstream and 1.7 kb downstream of the bop gene coding
region, so in strains SD16 and SD9 the bop probe hybridizes
with 3.5- and 4-kb fragments, respectively. All the transformants with
pXU3 and pXU10-15 that were analyzed had a single band of 15
kb hybridizing to the probe in the SmaI digests, indicating
that the entire plasmid was integrated into the chromosome at the bop locus.
DNA from the transformed SD16 and SD9 cells was also analyzed by digestion with BamHI and NotI. The BamHI site is located 388 bases upstream of the initiation codon, and the NotI site is located 16 bases upstream of the termination codon, so that the BamHI-NotI fragment containing the bop coding region is about 1.2 kb in the wild-type gene and the recombinant genes on the plasmids but 0.5-1.1 kb larger in the SD16 and SD9 chromosomes (with their ISH insertions). Southern blots consistently showed two bands (1.2 and 1.7 or 2.3 kb) hybridizing with a nick-translated bop gene probe. For the transformants with pXU10 and the other mutant bop genes, the question remained whether recombination between the plasmid and the chromosome had occurred upstream or downstream of the mutation, because the latter would result in regeneration of a wild-type gene in the chromosome. The blots were stripped and reprobed with an oligonucleotide specific for the mutated sequence in order to determine which one of the two genes had the mutation, the one inactivated with the insertion (the larger BamHI-NotI fragment) or the one with that could be transcribed and translated to make the protein (1.2-kb band).
In order to maximize the odds of obtaining transformants with functional mutant genes and inactivated wild-type genes, transformations with mutant bop genes were done using strain SD9. Because the ISH1 insertion is near the location of all the mutations introduced into the bop gene on the plasmids, recombination between the plasmid and the chromosome both upstream of the mutation (in the 5`-flanking region) or downstream of the mutation (and of the ISH1 insertion in the chromosome) would result in the mutant gene remaining intact. SD9 cells transformed with the various plasmids containing wild-type and mutant bop genes were analyzed using Southern blots of BamHI + NotI digests that were first probed with a random-primed bop gene and then stripped and reprobed with an oligonucleotide specific for the mutant sequence. In each case the smaller (1.2 kb) band containing the uninterrupted bop gene hybridized to the mutant probe, whereas the larger (2.3 kb) band containing the ISH1 insertion did not. This is shown for two transformants with pXU10A in Fig. 3. Transformations of SD16 with mutant bop genes, on the other hand, frequently resulted in regeneration of a wild-type bop gene.
Figure 3: Southern blot of DNA from mutant H. halobium strains digested with BamHI + NotI. A, The blot was probed with a random-primed bop gene fragment. B, the same blot was stripped and re-probed with an oligonucleotide specific for the mutant sequence in pXU10A. DNA was from SD9 cells transformed with the following plasmids: Lanes 1, pXU3 (wild-type bop gene); lanes 2, pWL102 (vector alone); lanes 3, pXU10; lanes 4 and 5, pXU10A.
In order to determine whether this lack of BR expression was due to lack of transcription of the bop gene with the deletion or lack of translation of the mRNA, RNA was isolated from XU10 and XU3 cells and analyzed by Northern blotting (Fig. 4). The bop mRNA in XU10 cells was found to be degraded to low molecular weight fragments that nevertheless still hybridized to the bop probe; the XU3 cells were always found to have only intact bop mRNA with the expected molecular weight as shown in Fig. 4.
Figure 4: Northern blots of RNA from H. halobium mutants. Lane 1, 1.2-kb BamHI-XbaI DNA fragment containing the bop gene, which was also labeled by random-priming and used as the probe for the blot; lanes 2-8, RNA isolated from the following H. halobium strains: lane 2, XU3; lane 3, XU15; lane 4, XU14; lane 5, XU13; lane 6, SD9 transformed with pWL102; lane 7, XU10; lane 8, XU10A.
Colonies and cultures of the three mutant strains appeared less purple than those of XU3 or S9. The amount of BR produced by strains XU13, XU14, and XU15 was compared with that of XU3, which has the same genetic background but no mutation in the bop gene, by looking at the spectra of membranes prepared from similar amounts of cells (Fig. 6). The spectra showed that XU13, XU14, and XU15 cells produced less than half as much BR as XU3. The experiment was repeated three times, and the relative amounts of BR (570 nm absorbance) found in strains XU13, XU14, and XU15 varied slightly but was consistently less than 50% of that in XU3 and distinctly more than in XU10A. The relative amounts of BR in the various strains was also compared by examining the intensities of the BO polypeptide bands when SDS-PAGE gels were run on the membranes (Fig. 7). The intensities of the bands on the SDS-PAGE gel shown in Fig. 7did not show much difference between XU13 and XU14, and the band for XU10A is barely detectable on the photograph. These results were consistent with the spectra taken on that set of membrane samples, which were not the ones used for Fig. 6. The gels also showed that the BR in all the strains, including that of XU13 with its altered leader peptide sequence, was fully processed to the mature form by removal of the 13 amino-terminal amino acids. The soluble fractions of the cells (not shown) did not contain any BR.
Figure 6: Spectra of membranes from H. halobium mutants. The relative absorbance at 568 nm (due to bacteriorhodopsin) gives an indication of the relative amounts of BR produced in each strain. Absorbances at 410 nm due to cytochromes and at 470-550 nm due to carotenoids are also visible. Spectra of membranes from strains XU3, XU15, XU13, XU14, XU10A, and SD9 (the parent strain) transformed with the vector pWL102 are shown.
Figure 7: SDS-PAGE gel of membranes from H. halobium mutants stained with Coomassie Blue. Lane 1, SD9 transformed with pWL102; lane 2, XU3; lane 3, XU13; lane 4, XU14; lane 5, XU10A.
Northern blots showed that the bop mRNA in XU13, XU14, and XU15 was the same size as in XU3 and was not degraded like that of XU10 (Fig. 4). The variations in intensity between the bands were probably due to differences in yield during RNA isolation and were reflected in the intensity of the total RNA staining with ethidium bromide in each lane on the gel used for the Northern blot (not shown).
XU10A cells were pink, in contrast to the orange SD9 and XU10 or the purple XU3 cultures. Northern blots showed no degradation for XU10A mRNA (Fig. 4). The spectrum of XU10A membranes showed a peak at 570 nm characteristic of BR, which appeared as a shoulder on the carotenoid peak similar to that in XU14 membranes (Fig. 6). A faint BR band could also be detected on SDS-PAGE gels (Fig. 7). The relative amounts of polypeptide detected on gels are consistent with the amounts of the chromophore detected in spectra, indicating that most or all of the BO produced had bound retinal to form BR, which produces the characteristic absorption spectrum. No BR could be detected in spectra nor gels run on the soluble fraction of XU10A cells (not shown).
Figure 8:
Pulse-chase of H. halobium strains with [S]methionine. SDS-PAGE gel
and PhosphorImager analysis of membranes from strains XU3, XU10A, and
XU14. Cells were pulsed for 2 h with
[
S]methionine and then chased for 0, 2, 18, 26,
and 42 h with unlabeled methionine.
Transformation of H. halobium SD16 and SD9 with the wild-type bop gene fully restored expression of BR; this was not surprising, because the wild-type gene had been inserted back into the chromosome at the bop locus. Efficient expression of the wild-type bop gene in strain L33 (which has an ISH2 insertion in the bop coding region like SD16) using a similar plasmid has been reported previously by Needleman's group(21) . Their plasmid also contained the brp gene upstream of bop and was usually, but not always, found integrated into the chromosome(21) . The reason we never found transformants with plasmids containing a bop gene that had not integrated into the chromosome may be a loss of unintegrated plasmids during the prolonged incubation period without drug selection.
The lack of BR production in the leader sequence deletion mutant XU10 was most likely caused by the degradation of the mRNA and perhaps lack of translation of any remaining message due to deletion of the putative ribosome-binding site, which is in the region coding for the leader sequence (see Fig. 1and Fig. 2). This ribosome-binding site is downstream of the initiation codon, unlike those of eubacteria, which are upstream of the initiation codon but similarly complementary to the 3` end of the 16S rRNA. Secondary structure and ribosome binding at the 5` end of the bop mRNA apparently enhances the efficiency of translation by an unknown mechanism but does not interfere with the binding of complementary tRNAs to these bases when translation is initiated.
Strains XU14 and XU15 have only silent mutations and produce wild-type BR; their bop mRNA was produced in normal amounts and was not found degraded, so the reduced level of BR expression in these strains must be due to decreased translation of bop mRNA. In the case of XU13, the presence of an amino acid substitution that created a major difference in the net charge of the leader peptide did not cause any additional reduction in the amount of BR found compared with XU15. Both XU13 and XU15 have mRNA sequences that are predicted to have reduced binding to the 16S rRNA as well as reduced stability of the mRNA secondary structure. This perturbation of the hairpin secondary structure would not occur in XU14, but the level of BR in this mutant was on average comparable with that of XU13 and XU15. This suggests that the ribosome-binding sequence determines the level of translation and the exact secondary structure of the hairpin may not be critical for translation. The presence of a hairpin may, however, be required to prevent rapid mRNA degradation.
The results obtained with the modified leader sequence deletion
mutant XU10A indicate that the presence of a ribosome-binding sequence
and/or hairpin loop at the 5` end of the bop mRNA in XU10A was
sufficient to stabilize the mRNA and facilitate a modest level of
translation. The reason that the level of BR expression was
significantly less than that in XU3 may be because the new
ribosome-binding site and secondary structure are not very close
replicas of those in the wild-type bop gene. In fact, the
structure of XU10A mRNA is in some ways more similar to that of the hop gene mRNA (see Fig. 1). This gene, coding for the
protein halorhodopsin, is expressed at much lower levels than bop(22) . Consistent with this interpretation is the
observation that bop-hop fusions in which the 5` untranslated
region (including the promoter) of the bop gene is fused to
the hop coding region do not significantly increase the level
of hop expression, but fusions that include a portion of the bop coding region (and therefore the bop ribosome-binding site) result in a high level of production of
halorhodopsin (23) . ()Similarly, fusion of the bop promoter to the sopI (sensory opsin I) gene does
not significantly increase expression of sopI, but fusions
that also include the first 13 or 21 codons of the bop gene
result in much higher expression levels(24) .
The BR produced in XU10A was assembled with the retinal chromophore, and it was all found integrated into the H. halobium membrane. We were unable to extract it from the other membrane components by high salt washes or density gradient centrifugation. Therefore, the leader peptide must not be required for proper folding or membrane insertion. It may, however, facilitate these processes in wild-type cells (i.e. the leader peptide may have a role in the kinetics of protein folding or membrane insertion). In either case, the absence of the leader may result in misfolding or improper targeting of a substantial fraction of the newly synthesized polypeptide. Ribosomes translating the bop gene have been found bound to membranes and to a 7S RNA with a proposed signal recognition particle (SRP)-like function(4) ; these interactions may involve the leader sequence and enhance the efficiency of membrane insertion. An altered amino-terminal sequence might also result in increased turnover of the BR in the membrane, but this was not observed.
The pulse-chase results suggest that the level of BR expression is determined by the rate of synthesis. Unfortunately, the rate of incorporation of labeled methionine into BR in XU10A cells was too slow to allow pulses shorter than 2 h. Therefore one cannot rule out the possibility that much of the BO polypeptide in this strain is rapidly degraded before it has a chance to be integrated into the membrane, because it is inefficiently translocated or misfolds in the absence of the leader sequence.