scholarly journals Translocation of α-Synuclein Expressed in Escherichia coli

2007 ◽  
Vol 189 (7) ◽  
pp. 2777-2786 ◽  
Author(s):  
Guoping Ren ◽  
Xi Wang ◽  
Shufeng Hao ◽  
Hongyu Hu ◽  
Chih-chen Wang
Keyword(s):  
Escherichia Coli ◽  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Lewy Bodies ◽  
Signal Recognition ◽  
Inner Membrane ◽  
Dependent Pathway

ABSTRACT α-Synuclein is a major component of Lewy bodies in Parkinson's disease. Although no signal sequence is apparent, α-synuclein expressed in Escherichia coli is mostly located in the periplasm. The possibilities that α-synuclein translocated into the periplasm across the inner membrane by the SecA or the Tat targeting route identified in bacteria and that α-synuclein was released through MscL were excluded. The signal recognition particle-dependent pathway is involved in the translocation of α-synuclein. The C-terminal 99-to-140 portion of the α-synuclein molecule plays a signal-like role for its translocation into the periplasm, cooperating with the central 61-to-95 section. The N-terminal 1-to-60 region is not required for this translocation.

scholarly journals Dissecting the Translocase and Integrase Functions of the Escherichia coli Secyeg Translocon

2000 ◽  
Vol 150 (3) ◽  
pp. 689-694 ◽  
Author(s):  
Hans-Georg Koch ◽  
Matthias Müller
Keyword(s):  
Escherichia Coli ◽  
Membrane Proteins ◽  
Protein Delivery ◽  
Signal Recognition Particle ◽  
Membrane Vesicles ◽  
Biologically Active ◽  
Secretory Proteins ◽  
Signal Recognition ◽  
Inner Membrane ◽  
Independent Protein

Recent evidence suggests that in Escherichia coli, SecA/SecB and signal recognition particle (SRP) are constituents of two different pathways targeting secretory and inner membrane proteins to the SecYEG translocon of the plasma membrane. We now show that a secY mutation, which compromises a functional SecY–SecA interaction, does not impair the SRP-mediated integration of polytopic inner membrane proteins. Furthermore, under conditions in which the translocation of secretory proteins is strictly dependent on SecG for assisting SecA, the absence of SecG still allows polytopic membrane proteins to integrate at the wild-type level. These results indicate that SRP-dependent integration and SecA/SecB-mediated translocation do not only represent two independent protein delivery systems, but also remain mechanistically distinct processes even at the level of the membrane where they engage different domains of SecY and different components of the translocon. In addition, the experimental setup used here enabled us to demonstrate that SRP-dependent integration of a multispanning protein into membrane vesicles leads to a biologically active enzyme.

scholarly journals The DsbA Signal Sequence Directs Efficient, Cotranslational Export of Passenger Proteins to the Escherichia coli Periplasm via the Signal Recognition Particle Pathway

2003 ◽  
Vol 185 (19) ◽  
pp. 5706-5713 ◽  
Author(s):  
Clark F. Schierle ◽  
Mehmet Berkmen ◽  
Damon Huber ◽  
Carol Kumamoto ◽  
Dana Boyd ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gene Fusion ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Signal Recognition ◽  
Sensitive Assay ◽  
Thioredoxin 1 ◽  
Passenger Proteins ◽  
Native Signal Sequence ◽  
Slight Inhibition

ABSTRACT The Escherichia coli cytoplasmic protein thioredoxin 1 can be efficiently exported to the periplasmic space by the signal sequence of the DsbA protein (DsbAss) but not by the signal sequence of alkaline phosphatase (PhoA) or maltose binding protein (MBP). Using mutations of the signal recognition particle (SRP) pathway, we found that DsbAss directs thioredoxin 1 to the SRP export pathway. When DsbAss is fused to MBP, MBP also is directed to the SRP pathway. We show directly that the DsbAss-promoted export of MBP is largely cotranslational, in contrast to the mode of MBP export when the native signal sequence is utilized. However, both the export of thioredoxin 1 by DsbAss and the export of DsbA itself are quite sensitive to even the slight inhibition of SecA. These results suggest that SecA may be essential for both the slow posttranslational pathway and the SRP-dependent cotranslational pathway. Finally, probably because of its rapid folding in the cytoplasm, thioredoxin provides, along with gene fusion approaches, a sensitive assay system for signal sequences that utilize the SRP pathway.

scholarly journals Use of Thioredoxin as a Reporter To Identify a Subset of Escherichia coli Signal Sequences That Promote Signal Recognition Particle-Dependent Translocation

2005 ◽  
Vol 187 (9) ◽  
pp. 2983-2991 ◽  
Author(s):  
Damon Huber ◽  
Dana Boyd ◽  
Yu Xia ◽  
Michael H. Olma ◽  
Mark Gerstein ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Signal Recognition ◽  
Cytoplasmic Protein ◽  
Signal Sequences ◽  
Simple Measure ◽  
Cotranslational Translocation ◽  
Dependent Signal ◽  
Thioredoxin 1

ABSTRACT We have previously reported that the DsbA signal sequence promotes efficient, cotranslational translocation of the cytoplasmic protein thioredoxin-1 via the bacterial signal recognition particle (SRP) pathway. However, two commonly used signal sequences, those of PhoA and MalE, which promote export by a posttranslational mechanism, do not export thioredoxin. We proposed that this difference in efficiency of export was due to the rapid folding of thioredoxin in the cytoplasm; cotranslational export by the DsbA signal sequence avoids the problem of cytoplasmic folding (C. F. Schierle, M. Berkmen, D. Huber, C. Kumamoto, D. Boyd, and J. Beckwith, J. Bacteriol. 185 :5706-5713, 2003). Here, we use thioredoxin as a reporter to distinguish SRP-dependent from non-SRP-dependent cleavable signal sequences. We screened signal sequences exhibiting a range of hydrophobicity values based on a method that estimates hydrophobicity. Successive iterations of screening and refining the method defined a threshold hydrophobicity required for SRP recognition. While all of the SRP-dependent signal sequences identified were above this threshold, there were also a few signal sequences above the threshold that did not utilize the SRP pathway. These results suggest that a simple measure of the hydrophobicity of a signal sequence is an important but not a sufficient indicator for SRP recognition. In addition, by fusing a number of both classes of signal sequences to DsbA, we found that DsbA utilizes an SRP-dependent signal sequence to achieve efficient export to the periplasm. Our results suggest that those proteins found to be exported by SRP-dependent signal sequences may require this mode of export because of their tendency to fold rapidly in the cytoplasm.

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

2006 ◽  
Vol 189 (5) ◽  
pp. 1783-1793 ◽  
Author(s):  
Olivera Francetic ◽  
Nienke Buddelmeijer ◽  
Shawn Lewenza ◽  
Carol A. Kumamoto ◽  
Anthony P. Pugsley
Keyword(s):  
Signal Sequence ◽  
Klebsiella Oxytoca ◽  
Signal Recognition Particle ◽  
Secretion System ◽  
Membrane Targeting ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Signal Recognition ◽  
Inner Membrane ◽  
Type Ii Secretion System

ABSTRACT 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 β-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.

scholarly journals Physiological Basis for Conservation of the Signal Recognition Particle Targeting Pathway in Escherichia coli

2001 ◽  
Vol 183 (7) ◽  
pp. 2187-2197 ◽  
Author(s):  
Harris D. Bernstein ◽  
Janine B. Hyndman
Keyword(s):  
Escherichia Coli ◽  
Heat Shock ◽  
Heat Shock Response ◽  
Signal Recognition Particle ◽  
Growth Conditions ◽  
Signal Recognition ◽  
Physiological Basis ◽  
Inner Membrane ◽  
Shock Response ◽  
Shock Proteins

ABSTRACT The Escherichia coli signal recognition particle (SRP) is a ribonucleoprotein complex that targets nascent inner membrane proteins (IMPs) to transport sites in the inner membrane (IM). Since SRP depletion only partially inhibits IMP insertion under some growth conditions, however, it is not clear why the particle is absolutely essential for viability. Insights into this question emerged from experiments in which we analyzed the physiological consequences of reducing the intracellular concentration of SRP below the wild-type level. We found that even moderate SRP deficiencies that have little effect on cell growth led to the induction of a heat shock response. Genetic manipulations that suppress the heat shock response were lethal in SRP-deficient cells, indicating that the elevated synthesis of heat shock proteins plays an important role in maintaining cell viability. Although it is conceivable that the heat shock response serves to increase the capacity of cells to target IMPs via chaperone-based mechanisms, SRP-deficient cells did not show an increased dependence on either GroEL or DnaK. By contrast, the heat shock-regulated proteases Lon and ClpQ became essential for viability when SRP levels were reduced. These results suggest that the heat shock response protects SRP-deficient cells by increasing their capacity to degrade mislocalized IMPs. Consistent with this notion, a model IMP that was mislocalized in the cytoplasm as the result of SRP depletion appeared to be more stable in a Δlon ΔclpQ strain than in control cells. Taken together, the data provide direct evidence that SRP is essential in E. coli and possibly conserved throughout prokaryotic evolution as well partly because efficient IMP targeting prevents a toxic accumulation of aggregated proteins in the cytoplasm.

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