Recruitment of coat proteins to liposomes and peptidoliposomes.

Intracellular transport within the cell is generally mediated by membrane vesicles. Their formation is typically initiated by activation of small GTPases that then recruit cytosolic proteins to the membrane surface to form a coat, interact with cargo and accessory proteins, and deform the lipid bilayer to produce a transport vesicle. Liposomes proved to be a useful tool to study the molecular mechanisms of these processes in vitro.

Interaction of amphiphysins with AP-1 clathrin adaptors at the membrane.

The assembly of clathrin/AP (adaptor protein)-1-coated vesicles on the trans-Golgi network and endosomes is much less studied than that of clathrin/AP-2 vesicles at the plasma membrane for endocytosis. In vitro, the association of AP-1 with protein-free liposomes had been shown to require phosphoinositides, Arf1 (ADP-ribosylation factor 1)-GTP and additional cytosolic factor(s). We have purified an active fraction from brain cytosol and found it to contain amphiphysin 1 and 2 and endophilin A1, three proteins known to be involved in the formation of AP-2/clathrin coats at the plasma membrane.

Recruitment of coat proteins to peptidoliposomes.

Intracellular transport between compartments within the cell is generally mediated by membrane vesicles. Their formation is initiated by activation of small GTPases that then recruit cytosolic proteins to the membrane surface to form a coat, interact with cargo proteins, and deform the lipid bilayer. Liposomes proved to be a useful tool to study the molecular mechanisms of these processes in vitro.

Oligomerization and dissociation of AP-1 adaptors are regulated by cargo signals and by ArfGAP1-induced GTP hydrolysis.

The mechanism of AP-1/clathrin coat formation was analyzed using purified adaptor proteins and synthetic liposomes presenting tyrosine sorting signals. AP-1 adaptors recruited in the presence of Arf1.GTP and sorting signals were found to oligomerize to high-molecular-weight complexes even in the absence of clathrin.

In vitro formation of recycling vesicles from endosomes requires adaptor protein-1/clathrin and is regulated by rab4 and the connector rabaptin-5.

The involvement of clathrin and associated adaptor proteins in receptor recycling from endosomes back to the plasma membrane is controversial. We have used an in vitro assay to identify the molecular requirements for the formation of recycling vesicles. Cells expressing the asialoglycoprotein receptor H1, a typical recycling receptor, were surface biotinylated and then allowed to endocytose for 10 min.