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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 expression of membrane proteins. E. coli cells harboring a plasmid encoding arabinose-inducible M, NG, and/or Ffh were induced to overexpress the indicated proteins. The expression levels of the indicated proteins were analyzed by Western blotting (panels A, B and C, using anti-Ffh, LacY, LacY-GFP, LacZ, or anti-His tag antibodies) or by “in-gel” fluorescence (C). The expression levels of LacZ and LacY were quantified and are shown as a percentages of their expression levels in samples with empty vector. The experiments were repeated three times, and the results shown are representative, with standard deviations that did not exceed 10%. As a loading control, antibodies against the ribosomal protein L15 were used. (D) E. coli IY228 (expressing chromosomally encoded LacY-GFP) harboring plasmids encoding arabinose-inducible M domain, NG domain, or Ffh or carrying empty vector were grown overnight on nylon membranes covering an LB agar plate containing a linear concentration gradient of arabinose (0 to 0.02%). The fluorescence was recorded using Typhoon.
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f3: Effect of the Ffh M domain on expression of membrane proteins. E. coli cells harboring a plasmid encoding arabinose-inducible M, NG, and/or Ffh were induced to overexpress the indicated proteins. The expression levels of the indicated proteins were analyzed by Western blotting (panels A, B and C, using anti-Ffh, LacY, LacY-GFP, LacZ, or anti-His tag antibodies) or by “in-gel” fluorescence (C). The expression levels of LacZ and LacY were quantified and are shown as a percentages of their expression levels in samples with empty vector. The experiments were repeated three times, and the results shown are representative, with standard deviations that did not exceed 10%. As a loading control, antibodies against the ribosomal protein L15 were used. (D) E. coli IY228 (expressing chromosomally encoded LacY-GFP) harboring plasmids encoding arabinose-inducible M domain, NG domain, or Ffh or carrying empty vector were grown overnight on nylon membranes covering an LB agar plate containing a linear concentration gradient of arabinose (0 to 0.02%). The fluorescence was recorded using Typhoon.

Mentions: Ffh has two domains connected by a linker (reviewed in reference 24). The N-terminal NG domain is homologous to the NG domain of FtsY and harbors the GTP-binding and hydrolysis activity. The C-terminal M domain (methionine rich) is responsible for interaction with 4.5S RNA and hydrophobic nascent peptides that emerge from ribosomes (reviewed in reference 24). To identify which domain of Ffh is responsible for the observed inhibition of membrane protein expression, Ffh or its NG or M domain was expressed with or without a C-terminal 6-histidine tag in cells induced for expression of the membrane protein LacY or the cytosolic protein LacZ (as a control). Initially, the amounts of LacY and LacZ in each strain were examined by Western blotting, and the results show that the M domain has a selective inhibitory effect on the expression of LacY (Fig. 3A, panels below upper panel). Only a minor difference was observed in the expression of LacY in cells overexpressing the NG domain, even though the amount of NG was substantially larger than that of the M domain (Fig. 3A, upper panel). The results were essentially the same with M, NG, or Ffh lacking a 6-His tag (data not shown and Fig. 3B). Interestingly, although coexpression of 4.5S RNA had a putative stabilizing effect on the M domain (Fig. 3B), as shown previously for Ffh (25–27), this coexpression did not change the phenotype observed in cells expressing the M domain alone. As noted above, overexpression of 4.5S RNA alone did not decrease the expression of LacY (Fig. 1). The effect of the M domain was further evaluated by utilizing a LacY-GFP hybrid (Fig. 3C). The GFP moiety enabled detection of differential expression in vivo (Fig. 3C, top panel) and with the GFP fluorescence of LacY-GFP separated by SDS-PAGE (Fig. 3C, panel marked “LacY-GFP fluorescence”). Overall, the results observed with LacY-GFP were identical to those described for LacY (Fig. 3A). Cells expressing LacY-GFP were also utilized to examine if the effect of M domain expression is concentration dependent. Cells coexpressing LacY-GFP and the arabinose-regulated M domain, NG domain, or Ffh protein were plated on an arabinose concentration gradient. After growth, the plate was scanned for GFP fluorescence as a measure for LacY-GFP expression (Fig. 3D), and the results clearly show that the inhibitory effects of the M domain and Ffh are concentration dependent. In accordance with the results shown above (Fig. 3A and C), the expression of LacY-GFP in cells harboring empty vector or overexpressing NG remained much less affected by elevated arabinose concentrations (Fig. 3D). Finally, we tested the effect of the M domain on the expression of other membrane proteins (Fig. 4), and the results show that the M domain-dependent inhibition is not restricted to LacY and that cytoplasmic proteins are not affected. Together, these studies suggest that the inhibitory effect of Ffh on membrane protein expression seems to be mediated by its 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 expression of membrane proteins. E. coli cells harboring a plasmid encoding arabinose-inducible M, NG, and/or Ffh were induced to overexpress the indicated proteins. The expression levels of the indicated proteins were analyzed by Western blotting (panels A, B and C, using anti-Ffh, LacY, LacY-GFP, LacZ, or anti-His tag antibodies) or by “in-gel” fluorescence (C). The expression levels of LacZ and LacY were quantified and are shown as a percentages of their expression levels in samples with empty vector. The experiments were repeated three times, and the results shown are representative, with standard deviations that did not exceed 10%. As a loading control, antibodies against the ribosomal protein L15 were used. (D) E. coli IY228 (expressing chromosomally encoded LacY-GFP) harboring plasmids encoding arabinose-inducible M domain, NG domain, or Ffh or carrying empty vector were grown overnight on nylon membranes covering an LB agar plate containing a linear concentration gradient of arabinose (0 to 0.02%). The fluorescence was recorded using Typhoon.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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f3: Effect of the Ffh M domain on expression of membrane proteins. E. coli cells harboring a plasmid encoding arabinose-inducible M, NG, and/or Ffh were induced to overexpress the indicated proteins. The expression levels of the indicated proteins were analyzed by Western blotting (panels A, B and C, using anti-Ffh, LacY, LacY-GFP, LacZ, or anti-His tag antibodies) or by “in-gel” fluorescence (C). The expression levels of LacZ and LacY were quantified and are shown as a percentages of their expression levels in samples with empty vector. The experiments were repeated three times, and the results shown are representative, with standard deviations that did not exceed 10%. As a loading control, antibodies against the ribosomal protein L15 were used. (D) E. coli IY228 (expressing chromosomally encoded LacY-GFP) harboring plasmids encoding arabinose-inducible M domain, NG domain, or Ffh or carrying empty vector were grown overnight on nylon membranes covering an LB agar plate containing a linear concentration gradient of arabinose (0 to 0.02%). The fluorescence was recorded using Typhoon.
Mentions: Ffh has two domains connected by a linker (reviewed in reference 24). The N-terminal NG domain is homologous to the NG domain of FtsY and harbors the GTP-binding and hydrolysis activity. The C-terminal M domain (methionine rich) is responsible for interaction with 4.5S RNA and hydrophobic nascent peptides that emerge from ribosomes (reviewed in reference 24). To identify which domain of Ffh is responsible for the observed inhibition of membrane protein expression, Ffh or its NG or M domain was expressed with or without a C-terminal 6-histidine tag in cells induced for expression of the membrane protein LacY or the cytosolic protein LacZ (as a control). Initially, the amounts of LacY and LacZ in each strain were examined by Western blotting, and the results show that the M domain has a selective inhibitory effect on the expression of LacY (Fig. 3A, panels below upper panel). Only a minor difference was observed in the expression of LacY in cells overexpressing the NG domain, even though the amount of NG was substantially larger than that of the M domain (Fig. 3A, upper panel). The results were essentially the same with M, NG, or Ffh lacking a 6-His tag (data not shown and Fig. 3B). Interestingly, although coexpression of 4.5S RNA had a putative stabilizing effect on the M domain (Fig. 3B), as shown previously for Ffh (25–27), this coexpression did not change the phenotype observed in cells expressing the M domain alone. As noted above, overexpression of 4.5S RNA alone did not decrease the expression of LacY (Fig. 1). The effect of the M domain was further evaluated by utilizing a LacY-GFP hybrid (Fig. 3C). The GFP moiety enabled detection of differential expression in vivo (Fig. 3C, top panel) and with the GFP fluorescence of LacY-GFP separated by SDS-PAGE (Fig. 3C, panel marked “LacY-GFP fluorescence”). Overall, the results observed with LacY-GFP were identical to those described for LacY (Fig. 3A). Cells expressing LacY-GFP were also utilized to examine if the effect of M domain expression is concentration dependent. Cells coexpressing LacY-GFP and the arabinose-regulated M domain, NG domain, or Ffh protein were plated on an arabinose concentration gradient. After growth, the plate was scanned for GFP fluorescence as a measure for LacY-GFP expression (Fig. 3D), and the results clearly show that the inhibitory effects of the M domain and Ffh are concentration dependent. In accordance with the results shown above (Fig. 3A and C), the expression of LacY-GFP in cells harboring empty vector or overexpressing NG remained much less affected by elevated arabinose concentrations (Fig. 3D). Finally, we tested the effect of the M domain on the expression of other membrane proteins (Fig. 4), and the results show that the M domain-dependent inhibition is not restricted to LacY and that cytoplasmic proteins are not affected. Together, these studies suggest that the inhibitory effect of Ffh on membrane protein expression seems to be mediated by its 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