scholarly journals The Structure of Multiple Polypeptide Domains Determines the Signal Recognition Particle Targeting Requirement ofEscherichia coli Inner Membrane Proteins

1999 ◽  
Vol 181 (15) ◽  
pp. 4561-4567 ◽  
Author(s):  
John A. Newitt ◽  
Nancy D. Ulbrandt ◽  
Harris D. Bernstein
Keyword(s):  
Escherichia Coli ◽  
Alkaline Phosphatase ◽  
Membrane Proteins ◽  
Signal Recognition Particle ◽  
Protein Export ◽  
Signal Recognition ◽  
Inner Membrane ◽  
Periplasmic Domain ◽  
Membrane Spanning ◽  
Membrane Spanning Domains

ABSTRACT The signal recognition particle (SRP) targeting pathway is required for the efficient insertion of many polytopic inner membrane proteins (IMPs) into the Escherichia coli inner membrane, but in the absence of SRP protein export proceeds normally. To define the properties of IMPs that impose SRP dependence, we analyzed the targeting requirements of bitopic IMPs that are structurally intermediate between exported proteins and polytopic IMPs. We found that disruption of the SRP pathway inhibited the insertion of only a subset of bitopic IMPs. Studies on a model bitopic AcrB-alkaline phosphatase fusion protein (AcrB 265-AP) showed that the SRP requirement for efficient insertion correlated with the presence of a large periplasmic domain (P1). As previously reported, perturbation of the SRP pathway also affected the insertion of a polytopic AcrB-AP fusion. Even exhaustive SRP depletion, however, failed to block the insertion of any AcrB derivative by more than 50%. Taken together, these data suggest that many proteins that are normally targeted by SRP can utilize alternative targeting pathways and that the structure of both hydrophilic and membrane-spanning domains determines the degree to which the biogenesis of a protein is SRP dependent.

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 Peculiar Properties of DsbA in Its Export across the Escherichia coli Cytoplasmic Membrane

2005 ◽  
Vol 187 (12) ◽  
pp. 3997-4004 ◽  
Author(s):  
Nobuyuki Shimohata ◽  
Yoshinori Akiyama ◽  
Koreaki Ito
Keyword(s):  
Escherichia Coli ◽  
Cytoplasmic Membrane ◽  
Polyacrylamide Gel Electrophoresis ◽  
Signal Recognition Particle ◽  
Sodium Dodecyl ◽  
Signal Recognition ◽  
Late Step ◽  
Periplasmic Domain ◽  
Protein Disulfide ◽  
Translocation Factors

ABSTRACT Export of DsbA, a protein disulfide bond-introducing enzyme, across the Escherichia coli cytoplasmic membrane was studied with special reference to the effects of various mutations affecting translocation factors. It was noted that both the internalized precursor retaining the signal peptide and the periplasmic mature product fold rapidly into a protease-resistant structure and they exhibited anomalies in sodium dodecyl sulfate-polyacrylamide gel electrophoresis in that the former migrated faster than the latter. The precursor, once accumulated, was not exported posttranslationally. DsbA export depended on the SecY translocon, the SecA ATPase, and Ffh (signal recognition particle), but not on SecB. SecY mutations, such as secY39 and secY205, that severely impair translocation of a number of secretory substrates by interfering with SecA actions only insignificantly impaired the DsbA export. In contrast, secY125, affecting a periplasmic domain and impairing a late step of translocation, exerted strong export inhibition of both classes of proteins. These results suggest that DsbA uses not only the signal recognition particle targeting pathway but also a special route of translocation through the translocon, which is hence suggested to actively discriminate preproteins.

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 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.

scholarly journals The role of the Tim8p–Tim13p complex in a conserved import pathway for mitochondrial polytopic inner membrane proteins

2002 ◽  
Vol 158 (6) ◽  
pp. 1017-1027 ◽  
Author(s):  
Sean P. Curran ◽  
Danielle Leuenberger ◽  
Einhard Schmidt ◽  
Carla M. Koehler
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Intermembrane Space ◽  
Hydrophobic Membrane ◽  
Inner Membrane ◽  
Membrane Complex ◽  
Mitochondrial Carrier Family ◽  
Pass Through ◽  
Membrane Spanning ◽  
Membrane Spanning Domains

Tim23p is imported via the TIM (translocase of inner membrane)22 pathway for mitochondrial inner membrane proteins. In contrast to precursors with an NH2-terminal targeting presequence that are imported in a linear NH2-terminal manner, we show that Tim23p crosses the outer membrane as a loop before inserting into the inner membrane. The Tim8p–Tim13p complex facilitates translocation across the intermembrane space by binding to the membrane spanning domains as shown by Tim23p peptide scans with the purified Tim8p–Tim13p complex and crosslinking studies with Tim23p fusion constructs. The interaction between Tim23p and the Tim8p–Tim13p complex is not dependent on zinc, and the purified Tim8p–Tim13p complex does not coordinate zinc in the conserved twin CX3C motif. Instead, the cysteine residues seemingly form intramolecular disulfide linkages. Given that proteins of the mitochondrial carrier family also pass through the TOM (translocase of outer membrane) complex as a loop, our study suggests that this translocation mechanism may be conserved. Thus, polytopic inner membrane proteins, which lack an NH2-terminal targeting sequence, pass through the TOM complex as a loop followed by binding of the small Tim proteins to the hydrophobic membrane spanning domains.

scholarly journals Compensating complete loss of signal recognition particle during co-translational protein targeting by the translation speed and accuracy

2021 ◽  
Author(s):  
Liuqun Zhao ◽  
Gang Fu ◽  
Yanyan Cui ◽  
Zixiang Xu ◽  
Dawei Zhang
Keyword(s):  
Membrane Proteins ◽  
Protein Targeting ◽  
Time Window ◽  
Signal Recognition Particle ◽  
Protein Translation ◽  
Suppressor Mutation ◽  
Signal Recognition ◽  
Translation Rate ◽  
Inner Membrane ◽  
Speed And Accuracy

Signal recognition particle (SRP) is critical for delivering co-translational proteins to the bacterial inner membrane. Previously, we identified SRP suppressors in Escherichia coli that inhibit translation initiation and elongation, which provides an insight into the mechanism of bypassing the requirement of SRP. Suppressor mutations tend to be located in regions that govern protein translation under evolutionary pressure. To verify this hypothesis, we re-executed the suppressor screening of SRP. Here we isolated a novel SRP suppressor mutation located in the Shine-Dalgarno sequence of S10 operon, which partially offset the targeting defects of SRP-dependent proteins. We found that the suppressor mutation slowed the translation rate of proteins, especially of inner membrane proteins, which could compensate for the targeting defects of inner membrane proteins via extending the time window of protein targeting. Furthermore, the fidelity of translation was decreased in suppressor cells, suggesting that the quality control of translation was inactivated to provide a survival advantage under the loss of SRP stress. Our results demonstrate that the inefficient protein targeting due to SRP deletion can be rescued through modulating translational speed and accuracy.

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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