Faculty Opinions recommendation of Genetic screen yields mutations in genes encoding all known components of the Escherichia coli signal recognition particle pathway.

2001 ◽  
Author(s):  
Koreaki Ito
Keyword(s):  
Escherichia Coli ◽  
Signal Recognition Particle ◽  
Genetic Screen ◽  
Signal Recognition ◽  
Genes Encoding

scholarly journals Genetic Screen Yields Mutations in Genes Encoding All Known Components of the Escherichia coli Signal Recognition Particle Pathway

2002 ◽  
Vol 184 (1) ◽  
pp. 111-118 ◽  
Author(s):  
Hongping Tian ◽  
Jon Beckwith
Keyword(s):  
Escherichia Coli ◽  
Signal Recognition Particle ◽  
Genetic Screen ◽  
Signal Recognition ◽  
Protein Insertion ◽  
E Coli ◽  
Genes Encoding ◽  
Membrane Protein Insertion ◽  
4.5S Rna ◽  
Distinct Membrane

ABSTRACT We describe the further utilization of a genetic screen that identifies mutations defective in the assembly of proteins into the Escherichia coli cytoplasmic membrane. The screen yielded mutations in each of the known genes encoding components of the E. coli signal recognition particle pathway: ffh, ffs, and ftsY, which encode Ffh, 4.5S RNA, and FtsY, respectively. In addition, the screen yielded mutations in secM, which is involved in regulating levels of the SecA component of the bacterium’s protein export pathway. We used a sensitive assay involving biotinylation to show that all of the mutations caused defects in the membrane insertions of three topologically distinct membrane proteins, AcrB, MalF, and FtsQ. Among the mutations that resulted in membrane protein insertion defects, only the secM mutations also showed defects in the translocation of proteins into the E. coli periplasm. Genetic evidence suggests that the S382T alteration of Ffh affects the interaction between Ffh and 4.5S RNA.

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 Functional interaction of chloroplast SRP/FtsY with the ALB3 translocase in thylakoids

2003 ◽  
Vol 162 (7) ◽  
pp. 1245-1254 ◽  
Author(s):  
Misty Moore ◽  
Robyn L. Goforth ◽  
Hiroki Mori ◽  
Ralph Henry
Keyword(s):  
Escherichia Coli ◽  
Signal Recognition Particle ◽  
Signal Recognition ◽  
Functional Interaction ◽  
Transport Pathway ◽  
Affinity Tags ◽  
Chl A ◽  
Target Membrane ◽  
Membrane Components ◽  
Subsequent Integration

Integration of thylakoid proteins by the chloroplast signal recognition particle (cpSRP) posttranslational transport pathway requires the cpSRP, an SRP receptor homologue (cpFtsY), and the membrane protein ALB3. Similarly, Escherichia coli uses an SRP and FtsY to cotranslationally target membrane proteins to the SecYEG translocase, which contains an ALB3 homologue, YidC. In neither system are the interactions between soluble and membrane components well understood. We show that complexes containing cpSRP, cpFtsY, and ALB3 can be precipitated using affinity tags on cpSRP or cpFtsY. Stabilization of this complex with GMP-PNP specifically blocks subsequent integration of substrate (light harvesting chl a/b-binding protein [LHCP]), indicating that the complex occupies functional ALB3 translocation sites. Surprisingly, neither substrate nor cpSRP43, a component of cpSRP, was necessary to form a complex with ALB3. Complexes also contained cpSecY, but its removal did not inhibit ALB3 function. Furthermore, antibody bound to ALB3 prevented ALB3 association with cpSRP and cpFtsY and inhibited LHCP integration suggesting that a complex containing cpSRP, cpFtsY, and ALB3 must form for proper LHCP integration.

A signal recognition particle in Escherichia coli?

1993 ◽  
Vol 3 (2) ◽  
pp. 86-89 ◽  
Author(s):  
Franz-Ulrich Hartl ◽  
Martin Wiedmann
Keyword(s):  
Escherichia Coli ◽  
Signal Recognition Particle ◽  
Signal Recognition

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 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.

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