Position-dependent Effects of Polylysine on Sec Protein Transport

Liang, Fu‐Cheng; Bageshwar, Umesh K.; Musser, Siegfried M.

doi:10.1074/jbc.m111.240903

Cited by 14 publications

(15 citation statements)

References 64 publications

(80 reference statements)

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“…Previous studies of translocation by SecYEG-SecA have shown that the ease with which a stretch of polypeptide chain passes through the substrate channel depends on the nature of the amino acids in that stretch ( Sato et al, 1997 ; Bauer et al, 2014 ). For example, poly-glycine regions diffuse freely and rapidly backwards and forth ( Bauer et al, 2014 ), while charged amino acids, especially positively charged, are excluded ( Nouwen et al, 2009 ; Liang et al, 2012 ). It has also been shown that some combinations of amino acids form secondary structural elements even before they exit the ribosome ( Hardesty and Kramer, 2001 ; Lu and Deutsch, 2005 ), while others remain natively unfolded.…”

Section: Resultsmentioning

confidence: 99%

Two-way communication between SecY and SecA suggests a Brownian ratchet mechanism for protein translocation

Allen

Corey

Oatley

et al. 2016

eLife

158

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The essential process of protein secretion is achieved by the ubiquitous Sec machinery. In prokaryotes, the drive for translocation comes from ATP hydrolysis by the cytosolic motor-protein SecA, in concert with the proton motive force (PMF). However, the mechanism through which ATP hydrolysis by SecA is coupled to directional movement through SecYEG is unclear. Here, we combine all-atom molecular dynamics (MD) simulations with single molecule FRET and biochemical assays. We show that ATP binding by SecA causes opening of the SecY-channel at long range, while substrates at the SecY-channel entrance feed back to regulate nucleotide exchange by SecA. This two-way communication suggests a new, unifying 'Brownian ratchet' mechanism, whereby ATP binding and hydrolysis bias the direction of polypeptide diffusion. The model represents a solution to the problem of transporting inherently variable substrates such as polypeptides, and may underlie mechanisms of other motors that translocate proteins and nucleic acids.DOI: http://dx.doi.org/10.7554/eLife.15598.001

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Section: Resultsmentioning

confidence: 99%

Two-way communication between SecY and SecA suggests a Brownian ratchet mechanism for protein translocation

Allen

Corey

Oatley

et al. 2016

eLife

158

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“…The neutral tags for P‐N5‐CalB and P‐Q5‐CalB also improved the extracellular production of CalB, but their extents were lower than the negative‐charged tags. The basic lysine tag prevented CalB expression in both the intracellular and extracellular spaces, of which reason was probably that poly lysine segments inhibited protein transport through the SecYEG protein translocation channel by electrostatic interaction (Liang et al, ). The highest specific lipase activities of 2.2 ± 0.7 U/mg cell in the culture broth and 0.3 ± 0.08 U/mg cell in the intracellular fraction were obtained for recombinant E. coli BL21 star (DE3) overexpressing P‐D5‐CalB, which were 8.4‐ and 3.0‐times higher than the corresponding values of the case of CalB alone.…”

Section: Resultsmentioning

confidence: 99%

Simple amino acid tags improve both expression and secretion of Candida antarctica lipase B in recombinant Escherichia coli

Kim

Park

Lee

et al. 2014

Biotech & Bioengineering

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Escherichia coli is the best-established microbial host strain for production of proteins and chemicals, but has a weakness for not secreting high amounts of active heterologous proteins to the extracellular culture medium, of which origins belong to whether prokaryotes or eukaryotes. In this study, Candida antarctica lipase B (CalB), a popular eukaryotic enzyme which catalyzes a number of biochemical reactions and barely secreted extracellularly, was expressed functionally at a gram scale in culture medium by using a simple amino acid-tag system of E. coli. New fusion tag systems consisting of a pelB signal sequence and various anion amino acid tags facilitated both intracellular expression and extracellular secretion of CalB. Among them, the N-terminal five aspartate tag changed the quaternary structure of the dimeric CalB and allowed production of 1.9 g/L active CalB with 65 U/mL activity in culture medium, which exhibited the same enzymatic properties as the commercial CalB. This PelB-anion amino acid tag-based expression system for CalB can be extended to production of other industrial proteins hardly expressed and exported from E. coli, thereby increasing target protein concentrations and minimizing purification steps.

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“…41,43,52,53 The goal of the current study is not to investigate the detailed mechanism of the SecA motor action; rather, we aim to characterize the conformational dynamics and mechanisms associated with nascent-protein insertion into the translocon. We thus model only the most fundamental roles of SecA in the insertion process: providing confinement of the nascent protein at the cytosolic mouth of the translocon channel and enforcing sequential insertion of the nascent protein into the translocon.…”

Section: Methodsmentioning

confidence: 99%

Direct Simulation of Early-Stage Sec-Facilitated Protein Translocation

Zhang

Miller

2012

J. Am. Chem. Soc.

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Direct simulations reveal key mechanistic features of early-stage protein translocation and membrane integration via the Sec-translocon channel. We present a novel computational protocol that combines non-equilibrium growth of the nascent protein with microsecond-timescale molecular dynamics trajectories. Analysis of multiple, long-timescale simulations elucidates molecular features of protein insertion into the translocon, including signal-peptide docking at the translocon lateral gate (LG), large-lengthscale conformational rearrangement of the translocon LG helices, and partial membrane integration of hydrophobic nascent-protein sequences. Furthermore, the simulations demonstrate the role of specific molecular interactions in the regulation of protein secretion, membrane integration, and integral membrane protein topology. Salt-bridge contacts between the nascent-protein N-terminus, cytosolic translocon residues, and phospholipid head groups are shown to favor conformations of the nascent protein upon early-stage insertion that are consistent with the Type II (Ncyt/Cexo) integral membrane protein topology; and extended hydrophobic contacts between the nascent protein and the membrane lipid bilayer are shown to stabilize configurations that are consistent with the Type III (Nexo/Ccyt) topology. These results provide a detailed, mechanistic basis for understanding experimentally observed correlations between integral membrane protein topology, translocon mutagenesis, and nascent-protein sequence.

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Position-dependent Effects of Polylysine on Sec Protein Transport

Cited by 14 publications

References 64 publications

Two-way communication between SecY and SecA suggests a Brownian ratchet mechanism for protein translocation

Two-way communication between SecY and SecA suggests a Brownian ratchet mechanism for protein translocation

Simple amino acid tags improve both expression and secretion of Candida antarctica lipase B in recombinant Escherichia coli

Direct Simulation of Early-Stage Sec-Facilitated Protein Translocation

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