Mapping polypeptide interactions of the SecA ATPase during translocation

Bauer, Benedikt; Rapoport, Tom A.

doi:10.1073/pnas.0910550106

Cited by 60 publications

(93 citation statements)

References 28 publications

(35 reference statements)

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“…The SecA amino terminus has been shown to interact with precursors, 5 lipids, 5,17,18 and SecYEG. 5,7 Here we have characterized the specific interaction between the SecA amino terminus and the C-terminal tails of SecB. It seems likely that the contact between SecA and the SecB tails must be broken so that the amino terminus of SecA is free to bind other partners along the pathway of transfer of polypeptides through the membrane.…”

Section: Discussionmentioning

confidence: 99%

“…[2][3][4] The surface on SecA that interacts with SecB overlaps with the surface that binds precursors as well as that which binds the SecYEG translocon. [5][6][7] Transfer of the ligand among these binding partners is a dynamic process in which the contacts between given proteins are likely to be replaced by contacts with other partners. To further elucidate this process, we have used isothermal titration calorimetry and analytical centrifugation to define the cognate binding site on SecB for the amino terminus of SecA.…”

Section: Introductionmentioning

confidence: 99%

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Direct identification of the site of binding on the chaperone SecB for the amino terminus of the translocon motor SecA

Randall

Henzl

2010

Protein Science

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Protein export mediated by the general secretory Sec system in Escherichia coli proceeds by a dynamic transfer of a precursor polypeptide from the chaperone SecB to the SecA ATPase motor of the translocon and subsequently into and through the channel of the membraneembedded SecYEG heterotrimer. The complex between SecA and SecB is stabilized by several separate sites of contact. Here we have demonstrated directly an interaction between the Nterminal residues 2 through 11 of SecA and the C-terminal 13 residues of SecB by isothermal titration calorimetry and analytical sedimentation velocity centrifugation. We discuss the unusual binding properties of SecA and SecB in context of a model for transfer of the precursor along the pathway of export.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Direct identification of the site of binding on the chaperone SecB for the amino terminus of the translocon motor SecA

Randall

Henzl

2010

Protein Science

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“…This finding has been confirmed by thiol-cross-linking (11,12) and by cryoelectron microscopy analysis of the eukaryotic Sec complex bound to a translating ribosome (13). It is thus well-established that a single SecY copy is sufficient to form the translocation pathway, yet it remains mysterious that the channel exists as oligomers (14)(15)(16)(17).…”

mentioning

confidence: 81%

Two copies of the SecY channel and acidic lipids are necessary to activate the SecA translocation ATPase

Dalal

Chan

Sligar

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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The SecA ATPase associates with the SecY complex to push preproteins across the bacterial membrane. Because a single SecY is sufficient to create the conducting channel, the function of SecY oligomerization remains unclear. Here, we have analyzed the translocation reaction using nanodiscs. We show that one SecY copy is sufficient to bind SecA and the preprotein, but only the SecY dimer together with acidic lipids supports the activation of the SecA translocation ATPase. In discs, the dimer is predominantly arranged in a back-to-back manner and remains active even if a constituent SecY copy is defective for SecA binding. In membrane vesicles and in intact cells, the coproduction of two inactive SecYs, one for channel gating and the other for SecA binding, recreates a functional translocation unit. These results indisputably argue that the SecY dimer is crucial for the activation of SecA, which is necessary for preprotein transport.

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“…35 S]methionine by in vitro translation with rabbit reticulocyte lysate (Promega) as described previously (Bauer and Rapoport, 2009).…”

Section: Bacterial In Vitro Translocation Assaymentioning

confidence: 99%

Decatransin, a novel natural product inhibiting protein translocation at the Sec61/SecY translocon

Junne

Wong

Studer

et al. 2015

Journal of Cell Science

Self Cite

116

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A new cyclic decadepsipeptide was isolated from Chaetosphaeria tulasneorum with potent bioactivity on mammalian and yeast cells. Chemogenomic profiling in S. cerevisiae indicated that the Sec61 translocon complex, the machinery for protein translocation and membrane insertion at the endoplasmic reticulum, is the target. The profiles were similar to those of cyclic heptadepsipeptides of a distinct chemotype (including HUN-7293 and cotransin) that had previously been shown to inhibit cotranslational translocation at the mammalian Sec61 translocon. Unbiased, genome-wide mutagenesis followed by full-genome sequencing in both fungal and mammalian cells identified dominant mutations in Sec61p (yeast) or Sec61a1 (mammals) that conferred resistance. Most, but not all, of these mutations affected inhibition by both chemotypes, despite an absence of structural similarity. Biochemical analysis confirmed inhibition of protein translocation into the endoplasmic reticulum of both co-and posttranslationally translocated substrates by both chemotypes, demonstrating a mechanism independent of a translating ribosome. Most interestingly, both chemotypes were found to also inhibit SecYEG, the bacterial Sec61 translocon homolog. We suggest 'decatransin' as the name for this new decadepsipeptide translocation inhibitor.

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Mapping polypeptide interactions of the SecA ATPase during translocation

Cited by 60 publications

References 28 publications

Direct identification of the site of binding on the chaperone SecB for the amino terminus of the translocon motor SecA

Direct identification of the site of binding on the chaperone SecB for the amino terminus of the translocon motor SecA

Two copies of the SecY channel and acidic lipids are necessary to activate the SecA translocation ATPase

Decatransin, a novel natural product inhibiting protein translocation at the Sec61/SecY translocon

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