Signal-sequence recognition by an Escherichia coli ribonucleoprotein complex

Luirink, Joen; High, Stephen; Wood, Heather E.; Giner, Angelika; Tollervey, David; Dobberstein, Bernhard

doi:10.1038/359741a0

Cited by 181 publications

(132 citation statements)

References 17 publications

Supporting

Mentioning

126

Contrasting

Order By: Relevance

“…Supernatant fractions were trichloroacetic acid-precipitated, washed with cold acetone, and resuspended in RN buffer. Samples were immunoprecipitated as described (19) or mixed directly with 2ϫ SDS-gel loading buffer before gel electrophoresis.…”

Section: Materials-restrictionmentioning

confidence: 99%

SecA Is Not Required for Signal Recognition Particle-mediated Targeting and Initial Membrane Insertion of a Nascent Inner Membrane Protein

Scotti¹,

Valent²,

Manting

et al. 1999

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

In Escherichia coli, signal recognition particle (SRP)-dependent targeting of inner membrane proteins has been described. In vitro cross-linking studies have demonstrated that short nascent chains exposing a highly hydrophobic targeting signal interact with the SRP. This SRP, assisted by its receptor, FtsY, mediates the transfer to a common translocation site in the inner membrane that contains SecA, SecG, and SecY. Here we describe a further in vitro reconstitution of SRP-mediated membrane insertion in which purified ribosomenascent chain-SRP complexes are targeted to the purified SecYEG complex contained in proteoliposomes in a process that requires the SRP-receptor FtsY and GTP. We found that in this system SecA and ATP are dispensable for both the transfer of the nascent inner membrane protein FtsQ to SecY and its stable membrane insertion. Release of the SRP from nascent FtsQ also occurred in the absence of SecYEG complex indicating a functional interaction of FtsY with lipids. These data suggest that SRP/FtsY and SecB/SecA constitute distinct targeting routes.Going across or integrating into the inner membrane presents two different challenges to proteins synthesized in the cytosol of a prokaryotic cell, and this is reflected by the existence of two main targeting routes. The SecB pathway is specialized for the targeting of periplasmic and outer membrane proteins. SecB is a cytosolic chaperone that binds to the mature region of a subset of preproteins (1). The SecB-preprotein complex is targeted to SecA, which is bound with high affinity to the membrane-embedded SecYEG complex (for review, see Ref.2).Both in vivo and in vitro experiments indicate that the signal recognition particle (SRP) 1 pathway is primarily used for the targeting of integral inner membrane proteins (3-7). This pathway resembles SRP-mediated targeting of proteins to the membrane of the endoplasmic reticulum (ER) in eukaryotes (for review, see Ref. 8). The eukaryotic SRP interacts with nascent membrane and secreted proteins and targets them to the SRP receptor SR␣ at the ER membrane. The eukaryotic SRP is a complex consisting of six proteins arranged on an RNA scaffold, the 7 S RNA. Escherichia coli contains a smaller SRP composed of the P48 protein and the 4.5 S RNA, which are homologous to the eukaryotic SRP54 and the 7 S RNA, respectively (for review, see Refs. 9 and 10). In addition, an SR␣ homologue has been identified in E. coli, designated FtsY (7, 11). Both P48 and FtsY (and their eukaryotic counterparts) are GTPases, and GTP binding and hydrolysis regulate the targeting cycle in a mechanism that has not yet been fully defined (12)(13)(14). In vivo overproduction of polytopic inner membrane proteins titrated out the limited amount of endogenous E. coli SRP (5), whereas depletion of essential SRP components affected membrane targeting of both polytopic and bitopic inner membrane proteins (3, 4).We have developed an in vitro cross-linking approach to dissect subsequent stages in SRP-mediated protein targeting. Short nascent inner m...

show abstract

Section: Materials-restrictionmentioning

confidence: 99%

SecA Is Not Required for Signal Recognition Particle-mediated Targeting and Initial Membrane Insertion of a Nascent Inner Membrane Protein

Scotti¹,

Valent²,

Manting

et al. 1999

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…Bifunctional cross-linking was induced with 1 mM bis(sulfosuccinimidyl) suberate (BS 3 ) for 10 min at 26°C and quenched at 4°C by adding 1͞10 volume of quench buffer (1 M glycine͞100 mM NaHCO 3 , pH 8.5). Ribosome-nascent chain complexes were collected as described (20) and analyzed either directly by SDS͞15% PAGE or after immunoprecipitation as described (21), using 4-fold the amount used for direct analysis. The [ 35 S]methionine-labeled bands were quantified by using IMAGEQUANT software (Molecular Dynamics) and corrected for translation efficiency of the nascent chains as described (20 …”

mentioning

confidence: 99%

SecB is a bona fide generalized chaperone in Escherichia coli

Ullers

Luirink

Harms

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

108

View full text Add to dashboard Cite

It is known that the DnaK and Trigger Factor (TF) chaperones cooperate in the folding of newly synthesized cytosolic proteins in Escherichia coli. We recently showed that despite a very narrow temperature range of growth and high levels of aggregated cytosolic proteins, E. coli can tolerate deletion of both chaperones, suggesting that other chaperones might be involved in this process. Here, we show that the secretion-dedicated chaperone SecB efficiently suppresses both the temperature sensitivity and the aggregation-prone phenotypes of a strain lacking both TF and DnaK. SecB suppression is independent of a productive interaction with the SecA subunit of the translocon. Furthermore, in vitro cross-linking experiments demonstrate that SecB can interact both co-and posttranslationally with short nascent chains of both secretory and cytosolic proteins. Finally, we show that such cotranslational substrate recognition by SecB is greatly suppressed in the presence of ribosome-bound TF, but not by DnaK. Taken together, our data demonstrate that SecB acts as a bona fide generalized chaperone.

show abstract

“…2.5 mM in water (90% H20, 10% D20) and [1,2]. SRP54 and Ffh are the units which bind directly with 1.5 mM in a mixed solvent of 50% TFE-da (Cambridge Isotope Labthe signal sequences [5,6]. From the sequence analysis, Bernoratories) and 50% unbuffered water (by volume).…”

Section: Introductionmentioning

confidence: 99%

Structure of a methionine‐rich segment of Escherichia coli Ffh protein

Chi

et al. 1996

FEBS Letters

View full text Add to dashboard Cite

. Materials and methods peptide assumes a stable a-helix along most of its length in aqueous TFE solution. It is clear that this segment of Ffh proteinThe Ffh(410-434) peptide was synthesized by the solid-phase methhas a very strong propensity to form a-helical structure, od on a MilliGen 9050 automated peptide synthesizer. The peptide was purified by reverse phase HPLC using a Phenomenex w-porex C8 Key words." Ffh protein; Peptide; NMR; CD; t~-Helix; column (150 mmx l0 mm, 10 gm). Elution was performed with a Translocation linear water-acetonitrile gradient containing 0.1% trifluoroacetic acid. The N-terminal sequence and molecular weight of the purified peptide were determined using an ABI protein sequencer and fast atom bombardment mass spectroscopy (JEOL JMS-HXll0/ll0A), respectively.

show abstract

Signal-sequence recognition by an Escherichia coli ribonucleoprotein complex

Cited by 181 publications

References 17 publications

SecA Is Not Required for Signal Recognition Particle-mediated Targeting and Initial Membrane Insertion of a Nascent Inner Membrane Protein

SecA Is Not Required for Signal Recognition Particle-mediated Targeting and Initial Membrane Insertion of a Nascent Inner Membrane Protein

SecB is a bona fide generalized chaperone in Escherichia coli

Structure of a methionine‐rich segment of Escherichia coli Ffh protein

Contact Info

Product

Resources

About