Kinetic Analysis of the Translocation of Fluorescent Precursor Proteins into Escherichia coli Membrane Vesicles

Keyzer, Jeanine de; Does, Chris van der; Driessen, Arnold J. M.

doi:10.1074/jbc.m208449200

Cited by 62 publications

(54 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The conditions employed in the in vitro assays represent multiple turnovers of the translocase (22). The experimental data show that 4 molecules of proOmpA-P1 (in a total of 1388 amino acids) are translocated in the same time as 1 molecule of proOmpA-P8 (in a total of 1397 amino acids).…”

Section: Discussionmentioning

confidence: 75%

“…tease protection assay followed by direct fluorescent detection in the gel (22). Quantitative analysis of the labeling efficiency indicated that all proOmpA derivatives were equally labeled with fluorescein maleimide, with an efficiency of ϳ45%.…”

Section: Construction and Purification Of Proompa Derivatives With Mumentioning

confidence: 99%

“…After purification, proteins were dialyzed against 50 mM bis-Tris propane, pH 7.4, 6 M urea (buffer B). ProOmpA-P1 and proOmpA derivatives were labeled with fluorescein maleimide (Molecular Probes) as described previously (22). Free label was removed by extensive dialysis against buffer B. Proteins were stored at Ϫ80°C.…”

Section: Introduction Of Multiple Periplasmic Domains Intomentioning

confidence: 99%

“…Reactions were stopped by chilling on ice. Non-translocated material was degraded by proteinase K treatment (11); thereafter, the translocated material was analyzed by SDS-PAGE followed by direct in gel visualization using a Roche Lumi Imager F1 (Roche Applied Science) (22). Translocation efficiency is expressed as percent of input protein.…”

Section: Introduction Of Multiple Periplasmic Domains Intomentioning

confidence: 99%

See 3 more Smart Citations

SecA Supports a Constant Rate of Preprotein Translocation

Tomkiewicz

Nouwen

Leeuwen

et al. 2006

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

In Escherichia coli, secretory proteins (preproteins) are translocated across the cytoplasmic membrane by the Sec system composed of a protein-conducting channel, SecYEG, and an ATP-dependent motor protein, SecA. After binding of the preprotein to SecYEG-bound SecA, cycles of ATP binding and hydrolysis by SecA are thought to drive the stepwise translocation of the preprotein across the membrane. To address how the length of a preprotein substrate affects the SecAdriven translocation process, we constructed derivatives of the precursor of the outer membrane protein A (proOmpA) with 2, 4, 6, and 8 in-tandem repeats of the periplasmic domain. With increasing polypeptide length, an increasing delay in the time before full-length translocation was observed, but the translocation rate expressed as amino acid translocation per minute remained constant. These data indicate that in the ATP-dependent reaction, SecA drives a constant rate of preprotein translocation consistent with a stepping mechanism of translocation.Translocation of secretory proteins (preproteins) across the cytoplasmic membrane of Escherichia coli is mediated by a multisubunit membraneembedded complex termed "translocase" (1). Translocase consists of a protein-conducting channel, SecYEG, and a peripheral ATPase, SecA. During or shortly after synthesis, the chaperone SecB binds the preprotein, maintains it in a translocation-competent state, and targets it to the SecYEGbound SecA (2, 3). Subsequently, the preprotein is translocated through the SecYEG pore using the energy from ATP hydrolysis and the proton-motive force (4 -6).In eukaryotes two hypotheses for the translocation of proteins across organellar membranes have been proposed: the "power-stroke" and the "molecular ratchet" mechanism (7-9). In prokaryotes the same mechanisms could drive the translocation of preproteins across the cytoplasmic membrane. In the power-stroke model, SecA would act as a forcegenerating motor; it binds to the preprotein, pushes it into the channel by binding of ATP, and then releases it in the channel upon the hydrolysis of ATP. Experimental evidence suggests that consecutive unfolded polypeptide segments of ϳ5 kDa are threaded through the SecYEG pore by cycles of ATP binding and hydrolysis at SecA (10 -12). However, it is not clear whether this fixed step size is maintained throughout the translocation reaction. This stepwise translocation model (or powerstroke model) predicts that the time required for the translocation of a preprotein is directly correlated to its length. Likewise, the amount of ATP required for translocation would be a linear function of the preprotein length. On the other hand, different factors may delay or accelerate translocation and the timing of the events. For instance, mildly hydrophobic polypeptide segments cause the accumulation of specific translocation intermediates that are stalled in the translocation pore (13). Such polypeptide segments likely slow down the overall translocation reaction.The second model, known as molecular ratchet-l...

show abstract

Section: Discussionmentioning

confidence: 75%

Section: Construction and Purification Of Proompa Derivatives With Mumentioning

confidence: 99%

Section: Introduction Of Multiple Periplasmic Domains Intomentioning

confidence: 99%

Section: Introduction Of Multiple Periplasmic Domains Intomentioning

confidence: 99%

See 2 more Smart Citations

SecA Supports a Constant Rate of Preprotein Translocation

Tomkiewicz

Nouwen

Leeuwen

et al. 2006

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…Association of SecA with the SecYEG translocon (26,30) promotes ADP release, and this step is closely coupled to translocation of precursor proteins (37,38). From kinetic data using MANT-ADP, it is evident that SecA2 releases ADP much more slowly than SecA1; consequently, the affinity of SecA2 for ADP is much higher than SecA1.…”

Section: Discussionmentioning

confidence: 97%

ADP-dependent Conformational Changes Distinguish Mycobacterium tuberculosis SecA2 from SecA1

D'Lima

Teschke

2014

Journal of Biological Chemistry

View full text Add to dashboard Cite

Background: Mycobacterium tuberculosis possesses a second SecA ATPase that is required for bacterial virulence. Results: ADP binding by SecA2 causes a conformational change observable by biochemical methods. Conclusion: ADP binding closes the clamp in SecA2. This is not observed in SecA1 or E. coli SecA. Significance: Nucleotide-dependent clamp closure suggests a mechanism by which mycobacterial SecA2 is distinguished from SecA1 by the translocation machinery.

show abstract

Oregon Green 488 maleimide

Sabnis¹

2015

Handbook of Fluorescent Dyes and Probes

View full text Add to dashboard Cite

Kinetic Analysis of the Translocation of Fluorescent Precursor Proteins into Escherichia coli Membrane Vesicles

Abstract: Protein secretion in

Cited by 62 publications

References 31 publications

SecA Supports a Constant Rate of Preprotein Translocation

SecA Supports a Constant Rate of Preprotein Translocation

ADP-dependent Conformational Changes Distinguish Mycobacterium tuberculosis SecA2 from SecA1

Oregon Green 488 maleimide

Contact Info

Product

Resources

About