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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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The Ffh M domain comigrates with the cytoplasmic ribosomes in a sucrose gradient. E. coli cells harboring a plasmid encoding arabinose-inducible M and carrying ffs were induced and harvested, and cell extracts were ultracentrifuged. The pellet fractions were loaded on top of a 10% to 30% (wt/vol) sucrose gradient containing either 10.5 mM MgCl2 (A) or 1 mM MgCl2 (B) and separated by ultracentrifugation for 70 min (A) or 105 min (B). The ODs (260 nm) of the fractions were measured (upper panels), and fractions were analyzed by Western blotting with antibodies against Ffh (and its M domain) (lower panels).
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f8: The Ffh M domain comigrates with the cytoplasmic ribosomes in a sucrose gradient. E. coli cells harboring a plasmid encoding arabinose-inducible M and carrying ffs were induced and harvested, and cell extracts were ultracentrifuged. The pellet fractions were loaded on top of a 10% to 30% (wt/vol) sucrose gradient containing either 10.5 mM MgCl2 (A) or 1 mM MgCl2 (B) and separated by ultracentrifugation for 70 min (A) or 105 min (B). The ODs (260 nm) of the fractions were measured (upper panels), and fractions were analyzed by Western blotting with antibodies against Ffh (and its M domain) (lower panels).

Mentions: The interaction between SRP and ribosomes in E. coli is well studied and characterized (21). Since translation inhibition might be mediated through direct interaction with the ribosome, we asked whether the M domain interacts with cytosolic ribosomes in our experimental system as shown previously (20). Cells transformed with a plasmid encoding the M domain were disrupted, and the ultracentrifuged pellets (including membranes and ribosomes) were separated by sucrose gradient centrifugation. Fractions were collected and examined for rRNA (A260) and M domain (Western blot) content. Figure 8A shows that free 70S ribosomes migrate in fractions 13 to 15 and that large portions of both the M domain and chromosomally expressed Ffh migrate in the same fractions. To distinguish between the possibility that the migration of the M domain at high sucrose density is due to aggregation and the possibility that this migration is due to association with the ribosomes, we utilized conditions under which ribosomes dissociate into their subunits, and the results show that both the M domain and Ffh now migrate in a lower density, primarily with the large (50s) subunit (Fig. 8B). These results suggest that Ffh might inhibit membrane protein expression via the direct interaction of its M domain (alone or with 4.5S RNA) with the large subunits of cytosolic ribosomes.


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)

The Ffh M domain comigrates with the cytoplasmic ribosomes in a sucrose gradient. E. coli cells harboring a plasmid encoding arabinose-inducible M and carrying ffs were induced and harvested, and cell extracts were ultracentrifuged. The pellet fractions were loaded on top of a 10% to 30% (wt/vol) sucrose gradient containing either 10.5 mM MgCl2 (A) or 1 mM MgCl2 (B) and separated by ultracentrifugation for 70 min (A) or 105 min (B). The ODs (260 nm) of the fractions were measured (upper panels), and fractions were analyzed by Western blotting with antibodies against Ffh (and its M domain) (lower panels).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f8: The Ffh M domain comigrates with the cytoplasmic ribosomes in a sucrose gradient. E. coli cells harboring a plasmid encoding arabinose-inducible M and carrying ffs were induced and harvested, and cell extracts were ultracentrifuged. The pellet fractions were loaded on top of a 10% to 30% (wt/vol) sucrose gradient containing either 10.5 mM MgCl2 (A) or 1 mM MgCl2 (B) and separated by ultracentrifugation for 70 min (A) or 105 min (B). The ODs (260 nm) of the fractions were measured (upper panels), and fractions were analyzed by Western blotting with antibodies against Ffh (and its M domain) (lower panels).
Mentions: The interaction between SRP and ribosomes in E. coli is well studied and characterized (21). Since translation inhibition might be mediated through direct interaction with the ribosome, we asked whether the M domain interacts with cytosolic ribosomes in our experimental system as shown previously (20). Cells transformed with a plasmid encoding the M domain were disrupted, and the ultracentrifuged pellets (including membranes and ribosomes) were separated by sucrose gradient centrifugation. Fractions were collected and examined for rRNA (A260) and M domain (Western blot) content. Figure 8A shows that free 70S ribosomes migrate in fractions 13 to 15 and that large portions of both the M domain and chromosomally expressed Ffh migrate in the same fractions. To distinguish between the possibility that the migration of the M domain at high sucrose density is due to aggregation and the possibility that this migration is due to association with the ribosomes, we utilized conditions under which ribosomes dissociate into their subunits, and the results show that both the M domain and Ffh now migrate in a lower density, primarily with the large (50s) subunit (Fig. 8B). These results suggest that Ffh might inhibit membrane protein expression via the direct interaction of its M domain (alone or with 4.5S RNA) with the large subunits of cytosolic ribosomes.

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