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Escherichia coli SRP, its protein subunit Ffh, and the Ffh M domain are able to selectively limit membrane protein expression when overexpressed.

Yosef I, Bochkareva ES, Bibi E - MBio (2010)

Bottom Line: The results show that SRP, Ffh, and the M domain are all able to selectively inhibit the expression of membrane proteins.We observed no apparent changes in the steady-state mRNA levels or membrane protein stability, suggesting that inhibition may occur at the level of translation, possibly through the interaction between Ffh and ribosome-hydrophobic nascent chain complexes.Since E. coli SRP does not have a eukaryote-like translation arrest domain, we discuss other possible mechanisms by which this SRP might regulate membrane protein translation when overexpressed.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel.

ABSTRACT
The Escherichia coli signal recognition particle (SRP) system plays an important role in membrane protein biogenesis. Previous studies have suggested indirectly that in addition to its role during the targeting of ribosomes translating membrane proteins to translocons, the SRP might also have a quality control role in preventing premature synthesis of membrane proteins in the cytoplasm. This proposal was studied here using cells simultaneously overexpressing various membrane proteins and either SRP, the SRP protein Ffh, its 4.5S RNA, or the Ffh M domain. The results show that SRP, Ffh, and the M domain are all able to selectively inhibit the expression of membrane proteins. We observed no apparent changes in the steady-state mRNA levels or membrane protein stability, suggesting that inhibition may occur at the level of translation, possibly through the interaction between Ffh and ribosome-hydrophobic nascent chain complexes. Since E. coli SRP does not have a eukaryote-like translation arrest domain, we discuss other possible mechanisms by which this SRP might regulate membrane protein translation when overexpressed.

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Effect of the Ffh M domain on the rate of synthesis of LacY-GFP in vivo. E. coli IY228 harboring plasmids encoding arabinose-inducible M and carrying ffs or empty vector were induced with arabinose, and then IPTG was added to induce expression of LacY-GFP. The cells were pulse-labeled with [35S]methionine and [35S]cysteine, and equal samples were withdrawn at the indicated time points. LacY-GFP was immunoprecipitated using anti-GFP antibodies. The precipitates were solubilized in 50 µl SDS sample buffer, and 25 µl was loaded on SDS-PAGE gel (12%), which was then dried and subjected to autoradiography. (B) The amount of labeled LacY-GFP was quantified by densitometry, and the averages of results from three independent assays are shown, with error bars representing standard deviations.
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f7: Effect of the Ffh M domain on the rate of synthesis of LacY-GFP in vivo. E. coli IY228 harboring plasmids encoding arabinose-inducible M and carrying ffs or empty vector were induced with arabinose, and then IPTG was added to induce expression of LacY-GFP. The cells were pulse-labeled with [35S]methionine and [35S]cysteine, and equal samples were withdrawn at the indicated time points. LacY-GFP was immunoprecipitated using anti-GFP antibodies. The precipitates were solubilized in 50 µl SDS sample buffer, and 25 µl was loaded on SDS-PAGE gel (12%), which was then dried and subjected to autoradiography. (B) The amount of labeled LacY-GFP was quantified by densitometry, and the averages of results from three independent assays are shown, with error bars representing standard deviations.

Mentions: How does the M domain regulate the expression of membrane proteins? To answer this question, we examined the effect of M domain overexpression on the stability and rate of synthesis of a membrane protein and the fate of its coding mRNA in cells harboring a chromosomally encoded LacY-GFP (Fig. 5; see also Fig. 7) or LacY (Fig. 6). Because of the stabilizing effect of 4.5S RNA on the M domain (Fig. 3B), we included 4.5S RNA coexpression in all the following experiments. The stability of LacY-GFP was investigated by pulse-chase experiments, and as expected, the results show that the level of LacY-GFP expression is substantially lower in cells expressing the M domain (Fig. 5A). Importantly, however, no difference in LacY-GFP stability was observed between cells overexpressing the M domain and control cells (Fig. 5B). Therefore, posttranslational degradation does not explain the low expression level of LacY in M domain-expressing cells. Next, quantitative real-time PCR studies were conducted with total RNA samples prepared from control cells and cells overexpressing the M domain. Figure 6 shows that the amounts of the LacY-encoding mRNA are similar in both cell types. We therefore suggest that the decreased expression of membrane proteins in Ffh- or M domain-overexpressing cells might be due to inhibition of translation. This notion is consistent with the results of a pulse experiment, where the kinetics of expression of LacY-GFP in cells overexpressing the M domain was measured (Fig. 7A and B). The results of this experiment clearly show that LacY synthesis is substantially more rapid in control cells than in those coexpressing the M domain.


Escherichia coli SRP, its protein subunit Ffh, and the Ffh M domain are able to selectively limit membrane protein expression when overexpressed.

Yosef I, Bochkareva ES, Bibi E - MBio (2010)

Effect of the Ffh M domain on the rate of synthesis of LacY-GFP in vivo. E. coli IY228 harboring plasmids encoding arabinose-inducible M and carrying ffs or empty vector were induced with arabinose, and then IPTG was added to induce expression of LacY-GFP. The cells were pulse-labeled with [35S]methionine and [35S]cysteine, and equal samples were withdrawn at the indicated time points. LacY-GFP was immunoprecipitated using anti-GFP antibodies. The precipitates were solubilized in 50 µl SDS sample buffer, and 25 µl was loaded on SDS-PAGE gel (12%), which was then dried and subjected to autoradiography. (B) The amount of labeled LacY-GFP was quantified by densitometry, and the averages of results from three independent assays are shown, with error bars representing standard deviations.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2921155&req=5

f7: Effect of the Ffh M domain on the rate of synthesis of LacY-GFP in vivo. E. coli IY228 harboring plasmids encoding arabinose-inducible M and carrying ffs or empty vector were induced with arabinose, and then IPTG was added to induce expression of LacY-GFP. The cells were pulse-labeled with [35S]methionine and [35S]cysteine, and equal samples were withdrawn at the indicated time points. LacY-GFP was immunoprecipitated using anti-GFP antibodies. The precipitates were solubilized in 50 µl SDS sample buffer, and 25 µl was loaded on SDS-PAGE gel (12%), which was then dried and subjected to autoradiography. (B) The amount of labeled LacY-GFP was quantified by densitometry, and the averages of results from three independent assays are shown, with error bars representing standard deviations.
Mentions: How does the M domain regulate the expression of membrane proteins? To answer this question, we examined the effect of M domain overexpression on the stability and rate of synthesis of a membrane protein and the fate of its coding mRNA in cells harboring a chromosomally encoded LacY-GFP (Fig. 5; see also Fig. 7) or LacY (Fig. 6). Because of the stabilizing effect of 4.5S RNA on the M domain (Fig. 3B), we included 4.5S RNA coexpression in all the following experiments. The stability of LacY-GFP was investigated by pulse-chase experiments, and as expected, the results show that the level of LacY-GFP expression is substantially lower in cells expressing the M domain (Fig. 5A). Importantly, however, no difference in LacY-GFP stability was observed between cells overexpressing the M domain and control cells (Fig. 5B). Therefore, posttranslational degradation does not explain the low expression level of LacY in M domain-expressing cells. Next, quantitative real-time PCR studies were conducted with total RNA samples prepared from control cells and cells overexpressing the M domain. Figure 6 shows that the amounts of the LacY-encoding mRNA are similar in both cell types. We therefore suggest that the decreased expression of membrane proteins in Ffh- or M domain-overexpressing cells might be due to inhibition of translation. This notion is consistent with the results of a pulse experiment, where the kinetics of expression of LacY-GFP in cells overexpressing the M domain was measured (Fig. 7A and B). The results of this experiment clearly show that LacY synthesis is substantially more rapid in control cells than in those coexpressing the M domain.

Bottom Line: The results show that SRP, Ffh, and the M domain are all able to selectively inhibit the expression of membrane proteins.We observed no apparent changes in the steady-state mRNA levels or membrane protein stability, suggesting that inhibition may occur at the level of translation, possibly through the interaction between Ffh and ribosome-hydrophobic nascent chain complexes.Since E. coli SRP does not have a eukaryote-like translation arrest domain, we discuss other possible mechanisms by which this SRP might regulate membrane protein translation when overexpressed.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel.

ABSTRACT
The Escherichia coli signal recognition particle (SRP) system plays an important role in membrane protein biogenesis. Previous studies have suggested indirectly that in addition to its role during the targeting of ribosomes translating membrane proteins to translocons, the SRP might also have a quality control role in preventing premature synthesis of membrane proteins in the cytoplasm. This proposal was studied here using cells simultaneously overexpressing various membrane proteins and either SRP, the SRP protein Ffh, its 4.5S RNA, or the Ffh M domain. The results show that SRP, Ffh, and the M domain are all able to selectively inhibit the expression of membrane proteins. We observed no apparent changes in the steady-state mRNA levels or membrane protein stability, suggesting that inhibition may occur at the level of translation, possibly through the interaction between Ffh and ribosome-hydrophobic nascent chain complexes. Since E. coli SRP does not have a eukaryote-like translation arrest domain, we discuss other possible mechanisms by which this SRP might regulate membrane protein translation when overexpressed.

Show MeSH
Related in: MedlinePlus