The E. coli Signal Recognition Particle Is Required for the Insertion of a Subset of Inner Membrane Proteins

Ulbrandt, Nancy D.; Newitt, John A.; Bernstein, Harris

doi:10.1016/s0092-8674(00)81839-5

Cited by 318 publications

(353 citation statements)

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“…When the AcrB fusion protein was expressed, all four strains bearing mutant Ffh-expressing plasmids grew significantly slower than wild-type controls (data not shown). These results are consistent with previous studies, which demonstrate that SRP targeting may be significantly impaired without substantially affecting cell growth (Ulbrandt et al, 1997). Reduced levels of active SRP in combination with overexpression of an SRP substrate such as AcrB, however, can lead to pronounced synthetically toxic effects (Ulbrandt et al, 1997;Bernstein and Hyndman, 2001).…”

supporting

confidence: 93%

“…These results are consistent with previous studies, which demonstrate that SRP targeting may be significantly impaired without substantially affecting cell growth (Ulbrandt et al, 1997). Reduced levels of active SRP in combination with overexpression of an SRP substrate such as AcrB, however, can lead to pronounced synthetically toxic effects (Ulbrandt et al, 1997;Bernstein and Hyndman, 2001). Western blotting of cell extracts with an Ffh-specific antibody showed that all Ffh variants were expressed to comparable levels ( Figure 5B, bottom).…”

supporting

confidence: 92%

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The Signal Recognition Particle (SRP) RNA Links Conformational Changes in the SRP to Protein Targeting

Bradshaw

Walter

2007

MBoC

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The RNA component of the signal recognition particle (SRP) is universally required for cotranslational protein targeting. Biochemical studies have shown that SRP RNA participates in the central step of protein targeting by catalyzing the interaction of the SRP with the SRP receptor (SR). SRP RNA also accelerates GTP hydrolysis in the SRP⅐SR complex once formed. Using a reverse-genetic and biochemical analysis, we identified mutations in the E. coli SRP protein, Ffh, that abrogate the activity of the SRP RNA and cause corresponding targeting defects in vivo. The mutations in Ffh that disrupt SRP RNA activity map to regions that undergo dramatic conformational changes during the targeting reaction, suggesting that the activity of the SRP RNA is linked to the major conformational changes in the signal sequence-binding subunit of the SRP. In this way, the SRP RNA may coordinate the interaction of the SRP and the SR with ribosome recruitment and transfer to the translocon, explaining why the SRP RNA is an indispensable component of the protein targeting machinery. INTRODUCTIONCotranslational protein targeting is a major route by which proteins are targeted to the membrane of the endoplasmic reticulum (or plasma membrane in prokaryotes). The machinery required for cotranslational protein targeting consists of the signal recognition particle (SRP), a protein/RNA complex that binds ribosomes translating secretory and membrane proteins, and the SRP receptor (SR), which resides at the membrane and binds to the SRP (Keenan et al., 2001). One of the highly conserved features of the targeting machinery is that SRP requires an RNA subunit to function (Walter and Blobel, 1982). This study elaborates the mechanism by which the SRP RNA subunit contributes to the protein targeting reaction.In the first step of cotranslational protein targeting, the SRP binds to the signal sequence of a nascent polypeptide chain emerging from the ribosome (Walter et al., 1981;Keenan et al., 2001;Halic et al., 2004). The resulting SRPribosome-nascent chain complex then binds to the SRP receptor (SR), which resides at the target membrane (Gilmore et al., 1982a,b). The SRP and the SR associate with each other through related GTPase modules, but only when GTPbound (Miller et al., 1993;Egea et al., 2004;Focia et al., 2004). After transfer of the ribosome to the protein translocation channel (translocon), the SRP and the SR hydrolyze their respective bound GTPs, which causes them to dissociate, and allows for a new round of targeting (Connolly et al., 1991). The SRP and the SR reciprocally activate the other's GTPase and are therefore GTPase-activating proteins (GAPs) for each other (Powers and Walter, 1995).Although SRP-dependent protein targeting is conserved in all organisms, the prokaryotic system has the fewest components and is therefore the simplest (Poritz et al., 1990;Larsen and Zwieb, 1993). In Escherichia coli, the SRP consists of a single protein, Ffh, and a small RNA, the 4.5S RNA (Poritz et al., 1990). Ffh consists of two domains: the M d...

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supporting

confidence: 93%

supporting

confidence: 92%

The Signal Recognition Particle (SRP) RNA Links Conformational Changes in the SRP to Protein Targeting

Bradshaw

Walter

2007

MBoC

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“…Plasmid Construction-Plasmids pHL12, pJH28, and pTRC-ftsY (G385A) have been described previously (12,28). To construct plasmid pHL20, the dnaK gene was first amplified by PCR using the oligonucleotides 5Ј-TATTACAGAGCTCACAACCACATGATGA-3Ј and 5Ј-ATC-TCGTAATAAGCTTGCTTAGCCATCT-3Ј and pS368 (28) as a template.…”

Section: Methodsmentioning

confidence: 99%

Trigger Factor Retards Protein Export in Escherichia coli

Lee

Bernstein

2002

Journal of Biological Chemistry

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Trigger factor (TF) is a ribosome-associated protein that interacts with a wide variety of nascent polypeptides in Escherichia coli. Previous studies have indicated that TF cooperates with DnaK to facilitate protein folding, but the basis of this cooperation is unclear. In this study we monitored protein export in E. coli that lack or overproduce TF to obtain further insights into its function. Whereas inactivation of genes encoding most molecular chaperones (including dnaK) impairs protein export, inactivation of the TF gene accelerated protein export and suppressed the need for targeting factors to maintain the translocation competence of presecretory proteins. Furthermore, overproduction of TF (but not DnaK) markedly retarded protein export. Manipulation of TF levels produced similar effects on the export of a cytosolic enzyme fused to a signal peptide. The data strongly suggest that TF has a unique ability to sequester nascent polypeptides for a relatively prolonged period. Based on our results, we propose that TF and DnaK promote protein folding by distinct (but complementary) mechanisms.Protein biogenesis in Escherichia coli is aided by a diverse set of specialized factors that interact with nascent polypeptides chains. Several of these factors ("molecular chaperones") promote the folding of cytoplasmic proteins by binding promiscuously to exposed hydrophobic surfaces and preventing aggregation (reviewed in Ref.

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“…Because acidurance is dependent on the membraneembedded H+/ATPase (Kobayashi et aE., 1986;) and since Ffh is involved in the localization of membrane proteins (Degier et al, 1997;Ulbrandt et al, 1997), we assayed for H+/ATPase activity in mutant membranes, particularly under acidic growth conditions. The results of the H+/ATPase assays performed on membrane fractions of S. mutans parent strain JHl005 and acid-sensitive mutant AS17 are shown in Table 1.…”

Section: Mutant As17 Exhibits a Defect In The H+/atpase Activity At Amentioning

confidence: 99%

Streptococcus mutans ffh, a gene encoding a homologue of the 54 kDa subunit of the signal recognition particle, is involved in resistance to acid stress

et al. 1999

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The ability of Streptococcus mutans, a bacterial pathogen associated with dental caries, t o tolerate rapid drops in plaque pH (acidurance), is considered an important virulence factor. To study this trait, Tn917 mutants of 5. mutans strain JH1005 which display acid sensitivity have been isolated and partially characterized. In this paper, the characterization of one of these mutants, AS1 7, is reported. Preliminary sequence analysis revealed that the transposon insertion in AS17 occurred in the intergenic region of a two-gene locus which has been named sat for secretion and acid tolerance. This locus displays a high degree of homology t o the y/xM-ffh operon of Bacillus subtik The sat+ locus was cloned by complementation of a conditional Escherichia coli ffh mutant with an 5. mutans genomic library. Sequencing of the complementing clone identified the intact yIxM and #%i genes as well as a partial ORF with homology t o the proU/opuAC gene of B. subtilis which encodes the binding protein of the ProU/OpuA osmoregulated glycine betaine transport system. RNA dot blot experiments indicated steady-state levels of ffh mRNA in the mutant that were approximately eightfold lower compared to parental levels. This suggests a partial polar effect of the sat=l::Tn917 mutation on ffh expression. Upon acid shock (pH 5)# wild-type ffh mRNA levels were found t o increase approximately four-to eightfold compared to unstressed (pH 7.5) levels. Mutant levels remained unaltered under the same conditions. Experiments designed to investigate the origins of the acid-sensitivity of the mutant revealed a lack of an acid-adaptiveholerance response. Assays of proton-extruding ATPase (H+/ATPase) specific activity measured with purified membranes derived from acidAshocked AS17 showed twofold lower levels compared to the parent strain. Also, AS17 was found to be unable t o ferment sorbitol although it was able t o grow in glucose and a variety of other sugar substrates. These findings suggest that Ffh may be involved in the maintenance of a functional membrane protein composition during adaptation of S. mutans t o changing environmental conditions. Abbreviations: ATR, acid tolerance response; GSP, general secretory pathway; SRP, signal recognition particle. KeywordsThe CenBank accession number for the DNA sequence of the 3 kb DNA fragment containing the y/xM-ffh genes is U88582. INTRODUCTIONStreptococcus mutans plays a major role in the aetiology of human dental caries. This bacterium is able to demineralize dental enamel by generation of a localized acidic environment in plaque by rapid glycolysis of dietary sugars to lactic acid. In addition to its acidogenicity, S. mutans is able to tolerate exposure to continual and rapid cycles of acid shock. In vivo pH measurements have demonstrated that the pH of plaque can drop from pH 7.0 to values ranging from 3.0 to 4.0 in less than 20 min following the intake of carbohydrates (Imfeld & Lutz, 1980;Yamada et al., 1980;Mormann & Muhlemann, 1981 ; Schachtele & Harlander, 1984;Jensen & Wefel, 1989). E...

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The E. coli Signal Recognition Particle Is Required for the Insertion of a Subset of Inner Membrane Proteins

Cited by 318 publications

References 43 publications

The Signal Recognition Particle (SRP) RNA Links Conformational Changes in the SRP to Protein Targeting

The Signal Recognition Particle (SRP) RNA Links Conformational Changes in the SRP to Protein Targeting

Trigger Factor Retards Protein Export in Escherichia coli

Streptococcus mutans ffh, a gene encoding a homologue of the 54 kDa subunit of the signal recognition particle, is involved in resistance to acid stress

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