Reconstitution of the Coat Protein Complex II Induces Morphological Changes on Artificial Membranes

Abstract

In eukaryotic cells, the transport of cargo from the endoplasmic reticulum (ER) to the Golgi apparatus is conducted by protein-coated membrane vesicles. The stepwise formation of the coat protein complex II (COPII) on ER membranes is initiated by the small GTPase Sar1. Following GDP/GTP exchange, which is catalyzed by the guanine nucleotide exchange factor (GEF) Sec12, Sar1 undergoes a conformational change upon which an amphipathic helix inserts into the proximal leaflet of the ER membrane. Membrane-bound Sar1p subsequently recruits the inner and outer coat subcomplexes Sec23/Sec24 and Sec13/Sec31 to complete the COPII coat. This process leads to marked changes in membrane curvature. We apply in vitro reconstitution studies with purified yeast proteins on giant unilamellar vesicles (GUVs) as an artificial membrane system to examine the COPII vesicle formation process. Using confocal microscopy for visualization, we observe the formation of rigid tubes protruding from the GUVs under conditions where GTP hydrolysis is prevented. Cryo-electron microscopy shows tubules that apparently fail to fission into separate vesicles. Moreover, we can distinguish individual stages in the formation of the COPII coat on the bilayer. Altering the lipid composition, we observe different membrane morphologies. Our in vitro investigation of the COPII complex shows that GTP hydrolysis is not essential for binding of the coat to the membrane but may have a role in vesicle fission.