Electron microscopic visualization of asymmetric precursor translocation intermediates: SecA functions as a dimer

Area of Science:

• Molecular Biology
• Structural Biology
• Cellular Biology

Background:

• SecA is the ATPase motor driving protein translocation across the bacterial inner membrane via the SecYEG channel.
• Understanding the dynamic structural changes of the translocase during preprotein translocation is crucial for elucidating protein export mechanisms.

Purpose of the Study:

• To investigate the oligomeric states and structural organization of SecA and SecYEG during preprotein translocation.
• To visualize the structural dynamics of translocation intermediates using electron microscopy.

Main Methods:

• Electron microscopy (EM) was employed to visualize the structures of *Escherichia coli* Sec translocation intermediates.
• Immunogold labeling was used to map surface topological changes of translocase components.

Main Results:

• The translocase holoenzyme, comprising SecA and SecYEG, forms a symmetrical assembly on proteoliposomes.
• Translocation intermediate 31 (I(31)) exhibits asymmetry due to preprotein binding, while SecA remains dimeric in both holoenzyme and I(31) complexes.
• The preprotein channel entry point was localized to the center of the I(31) structures.

Conclusions:

• The preprotein itself induces asymmetry in the translocation complex.
• SecA maintains a dimeric state throughout the preprotein translocation process.
• These findings provide structural insights into the mechanism of bacterial protein translocation.