37 / 120
Liposomes, Exosomes, and Virosomes: From Modeling Complex
Membrane Processes to Medical Diagnostics and Drug Delivery
Wednesday Speaker Abstracts
32
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.