A mutant hunt for defects in membrane protein assembly yields mutations affecting the bacterial signal recognition particle and Sec machinery

Tian, Hongping; Boyd, Dana; Beckwith, Jon

doi:10.1073/pnas.090087297

Cited by 81 publications

(78 citation statements)

References 44 publications

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“…Other genetic (59) and biochemical (28,60) approaches identified the signal recognition particle (SRP) as a key component for IM protein biogenesis in E. coli. Recently, a modification of the MalF-LacZ selection produced mutants affected in all known components of the SRP pathway (ffh, ffs, and ftsY) (58). Therefore, we reasoned that the LacZ fusion approach should be sufficiently sensitive to identify factors required for PulG localization.…”

Section: Resultsmentioning

confidence: 99%

Signal Recognition Particle-Dependent Inner Membrane Targeting of the PulG Pseudopilin Component of a Type II Secretion System

et al. 2007

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The pseudopilin PulG is an essential component of the pullulanase-specific type II secretion system from Klebsiella oxytoca. PulG is the major subunit of a short, thin-filament pseudopilus, which presumably elongates and retracts in the periplasm, acting as a dynamic piston to promote pullulanase secretion. It has a signal sequence-like N-terminal segment that, according to studies with green and red fluorescent protein chimeras, anchors unassembled PulG in the inner membrane. We analyzed the early steps of PulG inner membrane targeting and insertion in Escherichia coli derivatives defective in different protein targeting and export factors. The ␤-galactosidase activity in strains producing a PulG-LacZ hybrid protein increased substantially when the dsbA, dsbB, or all sec genes tested except secB were compromised by mutations. To facilitate analysis of native PulG membrane insertion, a leader peptidase cleavage site was engineered downstream from the N-terminal transmembrane segment (PrePulG*). Unprocessed PrePulG* was detected in strains carrying mutations in secA, secY, secE, and secD genes, including some novel alleles of secY and secD. Furthermore, depletion of the Ffh component of the signal recognition particle (SRP) completely abolished PrePulG* processing, without affecting the Sec-dependent export of periplasmic MalE and RbsB proteins. Thus, PulG is cotranslationally targeted to the inner membrane Sec translocase by SRP.

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Section: Resultsmentioning

confidence: 99%

Signal Recognition Particle-Dependent Inner Membrane Targeting of the PulG Pseudopilin Component of a Type II Secretion System

et al. 2007

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“…This disulfide detector consists of ␤-galactosidase, normally a cytoplasmic protein with four thiol groups, fused to the inner membrane protein MalF (24). In the presence of functional DsbA, it is believed that non-native disulfide bonds are formed in ␤-galactosidase, causing it to be inactive (25). In the absence of a periplasmic disulfide oxidant, ␤-galactosidase retains its reduced thiol groups and can fold to its active form (25).…”

Section: The Dsbc Disulfide Isomerization Pathway Is Involved In Coppermentioning

confidence: 99%

“…In the presence of functional DsbA, it is believed that non-native disulfide bonds are formed in ␤-galactosidase, causing it to be inactive (25). In the absence of a periplasmic disulfide oxidant, ␤-galactosidase retains its reduced thiol groups and can fold to its active form (25). Consistent with previous work, JCB816, a wild-type strain expressing the MalF-LacZ fusion protein, was white on X-gal, indicating that ␤-galactosidase was inactive in this strain and thus contained non-native disulfides (TABLE THREE).…”

Section: The Dsbc Disulfide Isomerization Pathway Is Involved In Coppermentioning

confidence: 99%

Copper Stress Causes an in Vivo Requirement for the Escherichia coli Disulfide Isomerase DsbC

Hiniker

Collet

Bardwell

2005

Journal of Biological Chemistry

152

173

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In Escherichia coli, the periplasmic disulfide oxidoreductase DsbA is thought to be a powerful but nonspecific oxidant, joining cysteines together the moment they enter the periplasm. DsbC, the primary disulfide isomerase, likely resolves incorrect disulfides. Given the reliance of protein function on correct disulfide bonds, it is surprising that no phenotype has been established for null mutations in dsbC. Here we demonstrate that mutations in the entire DsbC disulfide isomerization pathway cause an increased sensitivity to the redox-active metal copper. We find that copper catalyzes periplasmic disulfide bond formation under aerobic conditions and that copper catalyzes the formation of disulfide-bonded oligomers in vitro, which DsbC can resolve. Our data suggest that the copper sensitivity of dsbC ؊ strains arises from the inability of the cell to rearrange copper-catalyzed non-native disulfides in the absence of functional DsbC. Absence of functional DsbA augments the deleterious effects of copper on a dsbC ؊ strain, even though the dsbA ؊ single mutant is unaffected by copper. This may indicate that DsbA successfully competes with copper and forms disulfide bonds more accurately than copper does. These findings lead us to a model in which DsbA may be significantly more accurate in disulfide oxidation than previously thought, and in which the primary role of DsbC may be to rearrange incorrect disulfide bonds that are formed during certain oxidative stresses.Most periplasmic Escherichia coli proteins contain at least two cysteine residues and many are stable and active only when these cysteines form their native disulfide bond pairings (1). In E. coli, a family of thioldisulfide oxidoreductases ensures that periplasmic and secreted proteins form correct disulfide bonds. DsbA is the primary disulfide oxidant in the periplasm. It rapidly donates its disulfide directly to substrate proteins and oxidizes them (2). DsbA is believed to act as a relatively nonspecific oxidant, joining any two cysteines that approach each other (3). A dsbA Ϫ strain shows several in vivo phenotypes, including attenuated virulence and loss of motility, because of the absence of disulfide bonds in proteins involved in these pathways (4, 5). DsbC, a second periplasmic thiol-disulfide oxidoreductase, appears to function as a disulfide isomerase both in vitro and in vivo. In vitro, DsbC has been shown to rearrange non-native disulfides in well studied isomerization substrates such as BPTI and RNase A (6, 7). In vivo, DsbC is required for full activity of a handful of proteins containing at least one non-consecutive disulfide bond (1). We have found that the periplasmic proteins RNase I (four disulfides, one non-consecutive) and MepA (three disulfides, two non-consecutive) require DsbC for their stability and, in the case of RNase I, in vivo activity (1). Berkmen et al. (3) recently showed that the folding of Agp (three consecutive disulfides) becomes DsbC-dependent with the introduction of a non-consecutive disulfide bond. These results s...

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“…Commonly used reporter proteins such as alkaline phosphatase and ␤-galactosidase, which had proved invaluable for the genetic dissection of the Sec pathway cannot be employed for the analysis of the Tat pathway (2,(22)(23)(24). Specifically, the former cannot fold within the bacterial cytoplasm thereby precluding Tat export (Ref.…”

mentioning

confidence: 99%

Genetic Analysis of the Twin Arginine Translocator Secretion Pathway in Bacteria

DeLisa

Samuelson

Palmer

et al. 2002

Journal of Biological Chemistry

126

127

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The twin arginine translocation (Tat) pathway of bacteria and plant chloroplasts mediates translocation of essentially folded proteins across the cytoplasmic membrane. The detailed understanding of the mechanism of protein targeting to the Tat pathway has been hampered by the lack of screening or selection systems suitable for genetic analysis. We report here the development of a highly quantitative protein reporter for genetic analysis of Tat-specific export. Specifically, export via the Tat pathway rescues green fluorescent protein (GFP) fused to an SsrA peptide from degradation by the cytoplasmic proteolytic ClpXP machinery. As a result, cellular fluorescence is determined by the amount of GFP in the periplasmic space. We used the GFP-SsrA reporter to isolate gain-of-function mutants of a Tatspecific leader peptide and for the genetic analysis of the "invariant" signature RR dipeptide motif. Flow cytometric screening of trimethylamine N-oxide reductase (TorA) leader peptide libraries resulted in isolation of six gain-of function mutants that conferred significantly higher steady-state levels of export relative to the wildtype TorA leader. All the gain-of-function mutations occurred within or near the (S/T)RRXFLK consensus motif, highlighting the significance of this region in interactions with the Tat export machinery. Randomization of the consensus RR dipeptide in the TorA leader revealed that a basic side chain (R/K) is required at the first position whereas the second position can also accept Gln and Asn in addition to basic amino acids. This result indicates that twin arginine translocation does not require the presence of an arginine dipeptide within the conserved sequence motif.

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A mutant hunt for defects in membrane protein assembly yields mutations affecting the bacterial signal recognition particle and Sec machinery

Cited by 81 publications

References 44 publications

Signal Recognition Particle-Dependent Inner Membrane Targeting of the PulG Pseudopilin Component of a Type II Secretion System

Signal Recognition Particle-Dependent Inner Membrane Targeting of the PulG Pseudopilin Component of a Type II Secretion System

Copper Stress Causes an in Vivo Requirement for the Escherichia coli Disulfide Isomerase DsbC

Genetic Analysis of the Twin Arginine Translocator Secretion Pathway in Bacteria

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