Direct Demonstration of ATP-dependent Release of SecA from a Translocating Preprotein by Surface Plasmon Resonance

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

doi:10.1074/jbc.m303490200

Cited by 40 publications

(48 citation statements)

References 42 publications

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“…This is followed by a conformational change that occurs upon ATP binding at the translocon, and concomitantly, polypeptide chain movement proceeds. Hydrolysis of ATP results in the membrane deinsertion of SecA and release of SecA from the membrane surface (57). Considering that the signal peptide is likely to be passed through the relay system from SecA to SecY as the preprotein is translocated, it is important to ask how the SecY-signal peptide interaction is affected by SecA and nucleotides.…”

Section: Resultsmentioning

confidence: 99%

Demonstration of a Specific Escherichia coli SecY−Signal Peptide Interaction

et al. 2004

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Protein translocation in Escherichia coli is initiated by the interaction of a preprotein with the membrane translocase composed of a motor protein, SecA ATPase, and a membrane-embedded channel, the SecYEG complex. The extent to which the signal peptide region of the preprotein plays a role in SecYEG interactions is unclear, in part because studies in this area typically employ the entire preprotein. Using a synthetic signal peptide harboring a photoaffinity label in its hydrophobic core, we examined this interaction with SecYEG in a detergent micellar environment. The signal peptide was found to specifically bind SecY in a saturable manner and at levels comparable to those that stimulate SecA ATPase activity. Chemical and proteolytic cleavage of cross-linked SecY and analysis of the signal peptide adducts indicate that the binding was primarily to regions of the protein containing transmembrane domains seven and two. The signal peptide-SecY interaction was affected by the presence of SecA and nucleotides in a manner consistent with the transfer of signal peptide to SecY upon nucleotide hydrolysis at SecA. † This research was supported in part by National Institutes of Health Grant GM37639 (to D.A.K. Protein transport across, or integration into, biological membranes is a vital cellular process (1-3). Components of the Sec translocon, the membrane pore through which presecretory proteins (or membrane proteins) achieve membrane translocation (or integration), are the most conserved transport constituents throughout the three kingdoms of life (4).In Escherichia coli, the essential components of the translocase (5) include the membraneassociated form of SecA (6, 7) and the polytopic membrane proteins SecY, SecE (homologues of the mammalian Sec61α, Sec61γ, and the yeast ER 1 Sec61p, Sss1p, respectively), and SecG (8, 9); the latter three proteins form a stable trimeric SecYEG complex (10). SecA is an ATPase that powers the membrane translocation of hydrophilic polypeptides by coupling ATP hydrolysis with protein movement via concomitant SecA membrane insertion and deinsertion cycles (11,12). SecY protein has 10 transmembrane (TM1-TM10), six cytosolic (C1-C6), and five periplasmic (P1-P5) domains (13), and it forms the core of the passageway for the translocating polypeptide chain (14 (35,36). Suppressor analysis of prlA mutations using dysfunctional LamB signal peptides revealed that the suppressor mutations clustered in distinct regions, and TM7 of SecY was proposed to function in signal sequence recognition (37). Yet no investigation has focused on the direct interaction between a signal peptide and the E. coli translocon. Consequently, it remains unclear whether the nascent polypeptide chain is merely translocating in close proximity to SecY, SecY performs simply a proofreading function (37), or SecY is more intimately involved in the specific recognition of the signal peptide. Wang et al. Page 2Biochemistry. Author manuscript; available in PMC 2011 April 29. NIH-PA Author ManuscriptNIH-PA Author Manuscr...

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Section: Resultsmentioning

confidence: 99%

Demonstration of a Specific Escherichia coli SecY−Signal Peptide Interaction

et al. 2004

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“…27 The dissociation rates of the Glu176 mutants do not differ significantly from cysteine-less SecYEG (Table 1), while the association rates of the mutants are slightly lower than that of cysteine-less SecYEG. The resulting changes in K D (k off /k on ) are therefore small.…”

Section: Glu176 In Tms4c Is Important For Functionality But Not For Bmentioning

confidence: 88%

“…To study the binding of SecA to the SecY(R357) mutants, we immobilized IMVs containing the mutant SecYEG complexes on a Biacore Pioneer L1 chip and determined the association and dissociation rates for SecA by surface plasmon resonance (SPR). 27 The dissociation rates of the Arg357 mutants did not differ significantly from those of cysteine-less SecYEG ( Table 1). The association rate of SecY(R357E) was slightly lower than that of cysteine-less SecYEG, resulting in a lower affinity as defined by k off /k on .…”

Section: Cytoplasmic Loop C5 Of Secy Is In Close Proximity Of Sece Anmentioning

confidence: 90%

“…SPR measurements were performed on a Biacore 2000 essentially as described 27 but Tris-HCl (pH 8.0) was replaced by Hepes/NaOH (pH 7.0) in all steps. Approximately 3200 response units of IMVs were loaded on a Biacore Pioneer L1 chip, and SecA was injected at a (dimeric) concentration of 50 nM with a flow speed of 20 μl/min.…”

Section: Surface Plasmon Resonance(spr)mentioning

confidence: 99%

“…Fitting of the association and dissociation rates from the corrected response curves was performed as described, and gave nearly identical values to those described for the previous setup. 27 …”

Section: Surface Plasmon Resonance(spr)mentioning

confidence: 99%

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Identification of Two Interaction Sites in SecY that Are Important for the Functional Interaction with SecA

Sluis¹,

Nouwen²,

Koch³

et al. 2006

Journal of Molecular Biology

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Surface plasmon resonance for high‐throughput ligand screening of membrane‐bound proteins

Maynard

Lindquist

Sutherland

et al. 2009

Biotechnology Journal

109

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Technology based on surface plasmon resonance (SPR) has allowed rapid, label-free characterization of protein-protein and protein-small molecule interactions, from quantitative measurements of binding kinetics and thermodynamics and concentrations in complex samples to epitope analysis. SPR has become the gold standard in industrial and academic settings, in which typically the interaction between a pair of soluble binding partners is characterized in detail or a library of molecules is screened for binding against a single soluble protein. In spite of these successes, the technology is only beginning to be adapted to the needs of membrane-bound proteins. Including G protein-coupled receptors (GPCR), ion channels and other growth, immune and cellular receptors, these proteins are difficult to study in situ but represent promising targets for drug and biomarker development. Existing technologies, such as BIAcore™, have been adapted for membrane protein analysis by building supported lipid layers or vesicle capture on existing chips. Newer technologies, still in development, will allow membrane proteins to be presented in native or near-native formats. These include SPR nanopore arrays, in which lipid bilayers containing membrane proteins stably span small pores that are addressable from both sides of the bilayer. Here, we discuss successes with current SPR instrumentation and the potential for SPR nanopore arrays to enable quantitative, high-throughput screening of GPCR ligands, biomarker discovery involving membrane bound proteins and basic cellular biology.

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Direct Demonstration of ATP-dependent Release of SecA from a Translocating Preprotein by Surface Plasmon Resonance

Cited by 40 publications

References 42 publications

Demonstration of a Specific Escherichia coli SecY−Signal Peptide Interaction

Demonstration of a Specific Escherichia coli SecY−Signal Peptide Interaction

Identification of Two Interaction Sites in SecY that Are Important for the Functional Interaction with SecA

Surface plasmon resonance for high‐throughput ligand screening of membrane‐bound proteins

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