Biomedical Engineering Reference
In-Depth Information
Chapter 7
Recruitment of Coat Proteins to Liposomes
and Peptidoliposomes
Sonja Huser , Gregor Suri , Pascal Crottet , and Martin Spiess
Abstract
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.
Here we describe the use of liposomes and peptidoliposomes presenting lipid-coupled cytosolic tails of
cargo proteins for the in vitro analysis of the membrane recruitment of AP-1 adaptors in the process of
forming AP-1/clathrin coats. AP-1 recruitment is mediated by the GTPase Arf1 and requires specifi c lipids
and cargo signals. Interaction with cargo induces AP-1 oligomerization already in the absence of clathrin.
Without cargo peptides, accessory proteins, such as amphiphysin 2, can be identifi ed that stabilize AP-1
binding to liposomal membranes.
Key words
Amphiphysin, Arf1, Clathrin adaptor protein, Coat protein, Liposome, Membrane traffi c,
Peptidoliposome, Protein sorting
1
Introduction
Liposomes are widely used to study molecular processes at membrane
surfaces in vitro. They have been particularly useful in the area of
membrane traffi cking to reconstitute the assembly of cytosolic coat
proteins at the lipid bilayer and the formation of coated vesicles
from purifi ed components. Lipid and protein compositions, the
order of addition of individual components, and the conditions
(temperature, nucleotides, etc.) can be easily manipulated and help
to defi ne the minimal machinery of coat assembly and their molec-
ular mechanisms.
The three best characterized coats are coat protein (COP) I
mediating intra-Golgi and Golgi to endoplasmic reticulum (ER)
transport, COPII for vesicles derived from the ER, and clathrin
with various associated adaptor proteins (APs) for pathways
between the plasma membrane, endosomes, and the
trans
-Golgi
