Biophysical Society Thematic Meeting | Ascona, Switzerland

Liposomes, Exosomes, and Virosomes: From Modeling Complex Membrane Processes to Medical Diagnostics and Drug Delivery

Wednesday Speaker Abstracts

Reconstituted Membrane Insertion of Single Proteins in Real Time Andreas Kuhn . University of Hohenheim, Stuttgart, Baden-Württemberg, Germany.

The membrane insertase YidC inserts newly synthesized proteins into the plasma membrane. While defects in YidC homologs in animals and plants cause diseases, YidC in bacteria is essential for life. Membrane insertion and assembly of a functional ATP synthase and respiratory complexes is catalysed by the 6-spanning YidC protein of E. coli. To investigate how YidC interacts with the membrane-inserting proteins, we generated single cysteine mutants in YidC and in the model substrate, the Pf3 coat protein. The insertion process was then followed in a reconstituted system with purified components. The YidC protein was labeled with a fluorescent probe and assembled with lipids to form proteoliposomes. Single molecule FRET technology shows that the inserting Pf3 coat protein closely binds YidC at the cytoplasmic face of YidC with its C-terminal domain. Then, the N-terminal domain is translocated within milliseconds contacting the periplasmic domain of YidC at the outer face of the membrane. Observation of membrane-inserting complexes in vivo of single cysteine mutants of both the Pf3 coat protein and YidC was possible after coexpression and the analysis of disulfides formed during the first 30 sec of synthesis. The results show that the substrate contacts different YidC residues in 4 of the 6 transmembrane regions. The residues are located either in the region of the inner leaflet, in the membrane center as well in the periplasmic leaflet. These contacting sites show a consecutive transmembrane stack in the recently published crystal structure of YidC suggesting a substrate can slide in between TM3 and TM5 of YidC. A model will be presented how the Pf3 protein moves into the membrane bilayer by interaction with YidC.

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