Biophysical Society Thematic Meeting

Liposomes, Exosomes, and Virosomes: From Modeling Complex

Membrane Processes to Medical Diagnostics and Drug Delivery

Poster Abstracts

15-POS

Board 8

Biochemical and Structural Studies of FtsH, a Membrane Anchored Degradation Machine

Vanessa Carvalho

, Mohamed Chami

, Roland Kieffer

, Marie-Eve Aubin-Tam

, Henning

Stahlberg

, Andreas Engel

Delft University of Technology, Delft, Zuid Holland, Netherlands,

University of Basel, Basel,

Switzerland.

Proteases are responsible for elimination of non-functional proteins, controlling protein levels, or

modification of protein function. An important group of proteases are part of the ATPases

associated with various cellular activities proteases (AAA+) family. AAA+ proteases are

degradation machines, which exploit energy from ATP hydrolysis to unfold protein substrates

and translocate unfolded polypeptides through a central pore, down towards a degradation

chamber. In particular, FtsH is a membrane-anchored AAA+ protease, which play crucial roles

in membrane protein quality control, protein transport across the membrane and dislocation of

specific transmembrane segments. Although cytoplasmic structures are described, the full-length

structure and the route by which soluble or integral membrane proteins translocate into the FtsH

central pore to be unfolded, remains unclear. Structural characterization of full-length FtsH

solubilized with either detergent or styrene maleic acid (SMA) nanodiscs provides insights on

this mechanism and on FtsH integration in the lipid bilayer.

We optimise expression and purification protocols, using the full-length sequence that encodes

Aquifex Aeolicus

FtsH. The results of the detergent solubilized FtsH, in negative staining and

cryo-electron microscopy single particle analysis, show the first structure of the full-length FtsH.

Proteolytic and ATPase activities are also measured. We also use SMA as solubilisation agent,

which enables the formation of SMA-FtsH-nanodiscs such that FtsH remains in its native

membrane environment.