The Secretory Pathway From History to the State of the Art - Fusion of Biological Membranes and Related Problems - page 19

FIGURE 2. Model for a bivalent
interaction of coatomer with membrane-
bound ARF1-GTP and with the cyto-
plasmic domain of a p23 tetramer. Inter-
action with ARF1 occurs via a -COP

interface, and binding to p23 is mediated
via

β -/ γ

γ -COP.

4.2. Reconstitution of Coated Vesicles from Chemically
Defined Liposomes

In order to define the minimal requirements for the formation of COP-
coated vesicles, their budding was reconstituted from chemically defined
liposomes.
Using this reductionist system, it was shown that all that is needed to
form a COPI-coated vesicle are the cytosolic proteins ARF and coatomer
and the cytoplasmic domains of coat/cargo receptors (p24 family) emanat-
ing from the bilayer surface (Bremser et al., 1999). Liposome-derived
budding requires the presence of GTP and a temperature of 37°C similar
to what is observed for COPI vesicle formation from biological membranes
(Ostermann et al., 1993).This budding reaction was shown to be completely
independent of the lipid composition of donor liposomes provided that the
cytoplasmic tail of p23 (or another p24 family member) was present. As in
this reconstitution only the cytoplasmic domains of the p24 proteins are
essential, an interaction of p24 luminal domains with cargo proteins does
not seem to be necessary for coat assembly, consistent with earlier obser-
vations (Orci et al., 1986). COPI- (and COPII-) coated vesicle formation
from chemically defined liposomes without the need for any membrane
protein was reported depending on special lipid compositions (Matsuoka
et al., 1998; Spang et al ., 1998). However, this budding reaction was inde-
pendent on temperature and required a non-physiological composition of
phospholipids as well as the presence of a non-hydrolyzable analogue of
GTP. Thus, this reconstitution is likely to represent a partial event in
budding, bypassing other steps.
Taken together, reconstitution of COPI vesicle budding from artificial
lipid bilayers confirm that the molecular basis for assembly of a COPI
vesicle is provided by a bivalent interaction of coatomer to form a trimeric