title: SecA Cotranslationally Interacts with Nascent Substrate Proteins In Vivo.
abstract: 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.
authors: Institute for Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, United Kingdom d.huber@bham.ac.uk.|Center for Molecular Biology of Heidelberg University (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany.|German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors: Damon
authors: http://orcid.org/0000-0002-7367-3244
authors: D
authors: Huber
authors: Institute for Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, United Kingdom.
authors: Mohammed
authors: M
authors: Jamshad
authors: Institute for Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, United Kingdom.
authors: Ruby
authors: R
authors: Hanmer
authors: Center for Molecular Biology of Heidelberg University (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany.|German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors: Daniela
authors: D
authors: Schibich
authors: Center for Molecular Biology of Heidelberg University (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany.|German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors: Kristina
authors: K
authors: Döring
authors: Center for Molecular Biology of Heidelberg University (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany.|German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors: Isabella
authors: I
authors: Marcomini
authors: Center for Molecular Biology of Heidelberg University (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany.|German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors: Günter
authors: G
authors: Kramer
authors: Center for Molecular Biology of Heidelberg University (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany.|German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors: Bernd
authors: B
authors: Bukau
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D020411:Oligonucleotide Array Sequence Analysis; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D000069816:SEC Translocation Channels / Q000235:genetics / Q000378:metabolism*; D000081416:SecA Proteins
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012333:RNA, Messenger; D000069816:SEC Translocation Channels; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins
keywords: Sec; SecA; cotranslational translocation; posttranslational translocation; protein targeting; protein translocation; secretory pathway