scholarly journals Genetic Screen Yields Mutations in Genes Encoding All Known Components of the Escherichia coli Signal Recognition Particle Pathway

2002 ◽  
Vol 184 (1) ◽  
pp. 111-118 ◽  
Author(s):  
Hongping Tian ◽  
Jon Beckwith
Keyword(s):  
Escherichia Coli ◽  
Signal Recognition Particle ◽  
Genetic Screen ◽  
Signal Recognition ◽  
Protein Insertion ◽  
E Coli ◽  
Genes Encoding ◽  
Membrane Protein Insertion ◽  
4.5S Rna ◽  
Distinct Membrane

ABSTRACT We describe the further utilization of a genetic screen that identifies mutations defective in the assembly of proteins into the Escherichia coli cytoplasmic membrane. The screen yielded mutations in each of the known genes encoding components of the E. coli signal recognition particle pathway: ffh, ffs, and ftsY, which encode Ffh, 4.5S RNA, and FtsY, respectively. In addition, the screen yielded mutations in secM, which is involved in regulating levels of the SecA component of the bacterium’s protein export pathway. We used a sensitive assay involving biotinylation to show that all of the mutations caused defects in the membrane insertions of three topologically distinct membrane proteins, AcrB, MalF, and FtsQ. Among the mutations that resulted in membrane protein insertion defects, only the secM mutations also showed defects in the translocation of proteins into the E. coli periplasm. Genetic evidence suggests that the S382T alteration of Ffh affects the interaction between Ffh and 4.5S RNA.

scholarly journals SRP19 Is a Dispensable Component of the Signal Recognition Particle in Archaea

2006 ◽  
Vol 189 (1) ◽  
pp. 276-279 ◽  
Author(s):  
Sophie Yurist ◽  
Idit Dahan ◽  
Jerry Eichler
Keyword(s):  
Cell Growth ◽  
Membrane Protein ◽  
Protein Secretion ◽  
Signal Recognition Particle ◽  
Haloferax Volcanii ◽  
Signal Recognition ◽  
Protein Insertion ◽  
Membrane Protein Insertion ◽  
Srp Rna

ABSTRACT In vitro, archaeal SRP54 binds SRP RNA in the absence of SRP19, suggesting the latter to be expendable in Archaea. Accordingly, the Haloferax volcanii SRP19 gene was deleted. Although normally transcribed at a level comparable to that of the essential SRP54 gene, SRP19 deletion had no effect on cell growth, membrane protein insertion, protein secretion, or ribosome levels. The absence of SRP19 did, however, increase membrane bacterioruberin levels.

scholarly journals E. coli 4.5S RNA is part of a ribonucleoprotein particle that has properties related to signal recognition particle

Cell ◽  
1990 ◽  
Vol 63 (3) ◽  
pp. 591-600 ◽  
Author(s):  
Véronique Ribes ◽  
Karin Römisch ◽  
Angelika Giner ◽  
Bernhard Dobberstein ◽  
David Tollervey
Keyword(s):  
Signal Recognition Particle ◽  
Signal Recognition ◽  
Ribonucleoprotein Particle ◽  
E Coli ◽  
4.5S Rna

scholarly journals In Vitro Studies with Purified Components Reveal Signal Recognition Particle (SRP) and SecA/SecB as Constituents of Two Independent Protein-targeting Pathways of Escherichia coli

1999 ◽  
Vol 10 (7) ◽  
pp. 2163-2173 ◽  
Author(s):  
Hans-Georg Koch ◽  
Thomas Hengelage ◽  
Christoph Neumann-Haefelin ◽  
Juan MacFarlane ◽  
Hedda K. Hoffschulte ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Plasma Membrane ◽  
Membrane Proteins ◽  
Signal Recognition Particle ◽  
Membrane Vesicles ◽  
Secretory Proteins ◽  
Signal Recognition ◽  
Free System ◽  
E Coli

The molecular requirements for the translocation of secretory proteins across, and the integration of membrane proteins into, the plasma membrane of Escherichia coli were compared. This was achieved in a novel cell-free system from E. coliwhich, by extensive subfractionation, was simultaneously rendered deficient in SecA/SecB and the signal recognition particle (SRP) components, Ffh (P48), 4.5S RNA, and FtsY. The integration of two membrane proteins into inside-out plasma membrane vesicles of E. coli required all three SRP components and could not be driven by SecA, SecB, and ΔμH+. In contrast, these were the only components required for the translocation of secretory proteins into membrane vesicles, a process in which the SRP components were completely inactive. Our results, while confirming previous in vivo studies, provide the first in vitro evidence for the dependence of the integration of polytopic inner membrane proteins on SRP in E. coli. Furthermore, they suggest that SRP and SecA/SecB have different substrate specificities resulting in two separate targeting mechanisms for membrane and secretory proteins in E. coli. Both targeting pathways intersect at the translocation pore because they are equally affected by a blocked translocation channel.

An E. coli ribonucleoprotein containing 4.5S RNA resembles mammalian signal recognition particle

Science ◽  
1990 ◽  
Vol 250 (4984) ◽  
pp. 1111-1117 ◽  
Author(s):  
M. Poritz ◽  
H. Bernstein ◽  
K Strub ◽  
D Zopf ◽  
H Wilhelm ◽  
...  
Keyword(s):  
Signal Recognition Particle ◽  
Signal Recognition ◽  
E Coli ◽  
4.5S Rna

scholarly journals Functional Overlap but Lack of Complete Cross-Complementation of Streptococcus mutans and Escherichia coli YidC Orthologs

2008 ◽  
Vol 190 (7) ◽  
pp. 2458-2469 ◽  
Author(s):  
Yuxia Dong ◽  
Sara R. Palmer ◽  
Adnan Hasona ◽  
Shushi Nagamori ◽  
H. Ronald Kaback ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Streptococcus Mutans ◽  
Protein A ◽  
Membrane Integrity ◽  
Acid Tolerance ◽  
Growth Conditions ◽  
Stress Sensitivity ◽  
Protein Insertion ◽  
E Coli ◽  
Membrane Protein Insertion

ABSTRACT Oxa/YidC/Alb family proteins are chaperones involved in membrane protein insertion and assembly. Streptococcus mutans has two YidC paralogs. Elimination of yidC2, but not yidC1, results in stress sensitivity with decreased membrane-associated F1Fo ATPase activity and an inability to initiate growth at low pH or high salt concentrations (A. Hasona, P. J. Crowley, C. M. Levesque, R. W. Mair, D. G. Cvitkovitch, A. S. Bleiweis, and L. J. Brady, Proc. Natl. Acad. Sci. USA 102:17466-17471, 2005). We now show that Escherichia coli YidC complements for acid tolerance, and partially for salt tolerance, in S. mutans lacking yidC2 and that S. mutans YidC1 or YidC2 complements growth in liquid medium, restores the proton motive force, and functions to assemble the F1Fo ATPase in a previously engineered E. coli YidC depletion strain (J. C. Samuelson, M. Chen, F. Jiang, I. Moller, M. Wiedmann, A. Kuhn, G. J. Phillips, and R. E. Dalbey, Nature 406:637-641, 2000). Both YidC1 and YidC2 also promote membrane insertion of known YidC substrates in E. coli; however, complete membrane integrity is not fully replicated, as evidenced by induction of phage shock protein A. While both function to rescue E. coli growth in broth, a different result is observed on agar plates: growth of the YidC depletion strain is largely restored by 247YidC2, a hybrid S. mutans YidC2 fused to the YidC targeting region, but not by a similar chimera, 247YidC1, nor by YidC1 or YidC2. Simultaneous expression of YidC1 and YidC2 improves complementation on plates. This study demonstrates functional redundancy between YidC orthologs in gram-negative and gram-positive organisms but also highlights differences in their activity depending on growth conditions and species background, suggesting that the complete functional spectrum of each is optimized for the specific bacteria and environment in which they reside.

scholarly journals Secretion of LamB-LacZ by the Signal Recognition Particle Pathway of Escherichia coli

2003 ◽  
Vol 185 (19) ◽  
pp. 5697-5705 ◽  
Author(s):  
Christina Wilson Bowers ◽  
Fion Lau ◽  
Thomas J. Silhavy
Keyword(s):  
Escherichia Coli ◽  
Fusion Proteins ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Trigger Factor ◽  
Signal Recognition ◽  
E Coli ◽  
Secretion Pathway ◽  
Efficient Delivery

ABSTRACT LamB-LacZ fusion proteins have classically been used in studies of the general secretion pathway of Escherichia coli. Here we describe how increasing signal sequence hydrophobicity routes LamB-LacZ Hyb42-1 to the signal recognition particle (SRP) pathway. Secretion of this hydrophobic fusion variant (H*LamB-LacZ) was reduced in the absence of fully functional Ffh and Ffs, and the translocator jamming caused by Hyb42-1 was prevented by efficient delivery of the fusion to the periplasm. Finally, we found that in the absence of the ribosome-associated chaperone, trigger factor (Tig), LamB-LacZ localized to the periplasm in a SecA-dependent, SRP-independent fashion. Collectively, our results provide compelling in vivo evidence that there is an SRP-dependent cotranslational targeting mechanism in E. coli and argue against a role for trigger factor in pathway discrimination.

scholarly journals Ligand crowding at a nascent signal sequence

2003 ◽  
Vol 163 (1) ◽  
pp. 35-44 ◽  
Author(s):  
Gottfried Eisner ◽  
Hans-Georg Koch ◽  
Konstanze Beck ◽  
Joseph Brunner ◽  
Matthias Müller
Keyword(s):  
Escherichia Coli ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Secretory Protein ◽  
Trigger Factor ◽  
Cross Linking ◽  
Signal Recognition ◽  
Site Specific ◽  
E Coli ◽  
Nascent Chain

We have systematically analyzed the molecular environment of the signal sequence of a growing secretory protein from Escherichia coli using a stage- and site-specific cross-linking approach. Immediately after emerging from the ribosome, the signal sequence of pOmpA is accessible to Ffh, the protein component of the bacterial signal recognition particle, and to SecA, but it remains attached to the surface of the ribosome via protein L23. These contacts are lost upon further growth of the nascent chain, which brings the signal sequence into sole proximity to the chaperone Trigger factor (TF). In its absence, nascent pOmpA shows extended contacts with L23, and even long chains interact in these conditions proficiently with Ffh. Our results suggest that upon emergence from the ribosome, the signal sequence of an E. coli secretory protein gradually becomes sequestered by TF. Although TF thereby might control the accessibility of pOmpA's signal sequence to Ffh and SecA, it does not influence interaction of pOmpA with SecB.

scholarly journals Functional Analysis of the Signal Recognition Particle in Escherichia coli by Characterization of a Temperature-Sensitive ffh Mutant

2002 ◽  
Vol 184 (10) ◽  
pp. 2642-2653 ◽  
Author(s):  
Sei-Kyoung Park ◽  
Fenglei Jiang ◽  
Ross E. Dalbey ◽  
Gregory J. Phillips
Keyword(s):  
Escherichia Coli ◽  
Signal Recognition Particle ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Signal Recognition ◽  
E Coli ◽  
Elevated Expression ◽  
Type Gene ◽  
Shock Proteins

ABSTRACT The Ffh protein of Escherichia coli is a 48-kDa polypeptide that is homologous to the SRP54 subunit of the eukaryotic signal recognition particle (SRP). Efforts to understand the function of Ffh in bacteria have depended largely on the use of E. coli strains that allow depletion of the wild-type gene product. As an alternative approach to studying Ffh, a temperature-sensitive ffh mutant was isolated. The ffh-10(Ts) mutation results in two amino acid changes in conserved regions of the Ffh protein, and characterization of the mutant revealed that the cells rapidly lose viability at the nonpermissive temperature of 42°C as well as show reduced growth at the permissive temperature of 30°C. While the ffh mutant is defective in insertion of inner membrane proteins, the export of proteins with cleavable signal sequences is not impaired. The mutant also shows elevated expression of heat shock proteins and accumulates insoluble proteins, especially at 42°C. It was further observed that the temperature sensitivity of the ffh mutant was suppressed by overproduction of 4.5S RNA, the RNA component of the bacterial SRP, by stabilizing the thermolabile protein. Collectively, these results are consistent with a model in which Ffh is required only for localization of proteins integral to the cytoplasmic membrane and suggest new genetic approaches to the study of how the structure of the SRP contributes to its function.

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