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Related Concept Videos

Protein Translocation Machinery on the ER Membrane01:28

Protein Translocation Machinery on the ER Membrane

The translocon complex situated on the ER membrane is the main gateway for the protein secretory pathway. It facilitates the transport of nascent peptides into the ER lumen and their insertion into the ER membrane.
Sec61 protein conducting channel
In eukaryotes, the translocon complex comprises a core heterotrimeric translocator channel called the Sec61 complex. This channel includes three transmembrane proteins, Sec61α, Sec61β, and Sec61γ, and is the largest subunit of the translocon complex.
Cotranslational Protein Translocation01:20

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Post-translational Translocation of Proteins to the RER01:27

Post-translational Translocation of Proteins to the RER

A sizable fraction of proteins destined for ER are first synthesized in the cell cytosol and then transported across the ER membrane–a process called post-translational translocation. Similar to cotranslationally translocated proteins, these proteins also use the Sec translocon complex to enter the ER lumen.
Targeting proteins to the ER
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Bacterial protein secretion involves translocation systems to ensure proteins reach their designated locations, including the plasma membrane, periplasm, outer membrane, or the external environment. These translocation systems are vital for bacterial physiology, supporting processes like membrane assembly, enzymatic activity in the periplasm, and interactions with the external environment. The division of labor between Sec and Tat pathways ensures efficiency in handling proteins with diverse...

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Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies
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Published on: March 6, 2013

Preparation of a highly translocation-competent proOmpA/SecB complex.

Ken-Ichi Nishiyama1, Hajime Tokuda

  • 1Cryobiofrontier Research Center, Faculty of Agriculture, Iwate University, Morioka, Iwate 020-8550, Japan. nishiyam@iwate-u.ac.jp

Protein Science : a Publication of the Protein Society
|October 15, 2010
PubMed
Summary

Researchers developed methods to prepare translocation-competent proOmpA using SecB chaperone. This allows for efficient protein transport, overcoming jamming issues within the translocon for improved bacterial protein export.

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Area of Science:

  • Molecular Biology
  • Protein Translocation
  • Bacterial Protein Export

Background:

  • The proper folding and translocation of outer membrane proteins like proOmpA are crucial for bacterial cell envelope integrity.
  • Incompetent folding can lead to jamming within the protein translocation machinery (translocon).
  • SecB is a dedicated chaperone involved in targeting proteins to the Sec translocon.

Purpose of the Study:

  • To develop reproducible methods for preparing translocation-competent proOmpA.
  • To utilize SecB as a probe to differentiate between competent and incompetent forms of proOmpA.
  • To characterize the translocation competence of proOmpA/SecB complexes.

Main Methods:

  • Differential sorting of proOmpA using SecB as a probe.
  • Trypsin digestion to analyze protein folding states.
  • In vivo expression of proOmpA and SecB as a synthetic operon in E. coli.
  • In vitro synthesis using a continuous exchange cell-free system.
  • Purification of proOmpA/SecB complexes using His-tagged SecB.
  • Assay of translocation competence via SecG topology inversion.

Main Results:

  • An incompetent form of proOmpA was identified, characterized by partial N-terminal folding and jamming within the translocon.
  • SecB successfully differentiated between competent and incompetent proOmpA.
  • The proOmpA/SecB complex, generated both in vivo and in vitro, demonstrated high translocation competence, evidenced by SecG inversion.
  • Purified complexes retained activity despite sensitivity of the proOmpA signal peptide to proteolysis.

Conclusions:

  • SecB is an effective probe for identifying translocation-competent proOmpA.
  • The formation of a proOmpA/SecB complex is critical for overcoming translocation barriers.
  • Reproducible methods for generating translocation-competent proOmpA/SecB complexes were established, applicable both in vivo and in vitro.
  • These findings provide insights into bacterial protein export mechanisms and potential strategies for manipulating protein translocation.