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SecA functions in vivo as a discrete anti-parallel dimer to promote protein transport.

ABSTRACT: SecA ATPase motor protein plays a central role in bacterial protein transport by binding substrate proteins and the SecY channel complex and utilizing its ATPase activity to drive protein translocation across the plasma membrane. SecA has been shown to exist in a dynamic monomer-dimer equilibrium modulated by translocation ligands, and multiple structural forms of the dimer have been crystallized. Since the structural form of the dimer remains a controversial and unresolved question, we addressed this matter by engineering ?-benzoylphenylalanine along dimer interfaces corresponding to the five different SecA X-ray structures and assessing their in vivo photo-crosslinking pattern. A discrete anti-parallel 1M6N-like dimer was the dominant if not exclusive dimer found in vivo, whether SecA was cytosolic or in lipid or SecYEG-bound states. SecA bound to a stable translocation intermediate was crosslinked in vivo to a second SecA protomer at its 1M6N interface, suggesting that this specific dimer likely promotes active protein translocation. Taken together, our studies strengthen models that posit, at least in part, a SecA dimer-driven translocation mechanism.

SUBMITTER: Banerjee T

PROVIDER: S-EPMC5263173 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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SecA functions in vivo as a discrete anti-parallel dimer to promote protein transport.

Banerjee Tithi T Lindenthal Christine C Oliver Donald D

Molecular microbiology 20161207 3

SecA ATPase motor protein plays a central role in bacterial protein transport by binding substrate proteins and the SecY channel complex and utilizing its ATPase activity to drive protein translocation across the plasma membrane. SecA has been shown to exist in a dynamic monomer-dimer equilibrium modulated by translocation ligands, and multiple structural forms of the dimer have been crystallized. Since the structural form of the dimer remains a controversial and unresolved question, we addresse ...[more]

PMID: 27802584

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Project description:SecA is an essential component of the Sec machinery in bacteria, which is responsible for transporting proteins across the cytoplasmic membrane. Recent work from our laboratory indicates that SecA binds to ribosomes. Here, we used two different approaches to demonstrate that SecA also interacts with nascent polypeptides in vivo and that these polypeptides are Sec substrates. First, we photo-cross-linked SecA to ribosomes in vivo and identified mRNAs that copurify with SecA. Microarray analysis of the copurifying mRNAs indicated a strong enrichment for proteins containing Sec-targeting sequences. Second, we used a 2-dimensional (2-D) gel approach to analyze radioactively labeled nascent polypeptides that copurify with SecA, including maltose binding protein, a well-characterized SecA substrate. The interaction of SecA with nascent chains was not strongly affected in cells lacking SecB or trigger factor, both of which also interact with nascent Sec substrates. Indeed, the ability of SecB to interact with nascent chains was disrupted in strains in which the interaction between SecA and the ribosome was defective. Analysis of the interaction of SecA with purified ribosomes containing arrested nascent chains in vitro indicates that SecA can begin to interact with a variety of nascent chains when they reach a length of ∼110 amino acids, which is considerably shorter than the length required for interaction with SecB. Our results suggest that SecA cotranslationally recognizes nascent Sec substrates and that this recognition could be required for the efficient delivery of these proteins to the membrane-embedded Sec machinery. SecA is an ATPase that provides the energy for the translocation of proteins across the cytoplasmic membrane by the Sec machinery in bacteria. The translocation of most of these proteins is uncoupled from protein synthesis and is frequently described as "posttranslational." Here, we show that SecA interacts with nascent Sec substrates. This interaction is not dependent on SecB or trigger factor, which also interact with nascent Sec substrates. Moreover, the interaction of SecB with nascent polypeptides is dependent on the interaction of SecA with the ribosome, suggesting that interaction of the nascent chain with SecA precedes interaction with SecB. Our results suggest that SecA could recognize substrate proteins cotranslationally in order to efficiently target them for uncoupled protein translocation.

Dataset Information

SecA functions in vivo as a discrete anti-parallel dimer to promote protein transport.

Publications

SecA functions in vivo as a discrete anti-parallel dimer to promote protein transport.

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