Small G Proteins: Arf Family GTPases in Vesicular Transport - Ras Superfamily Small G Proteins: Biology and Mechanisms 2 - page 188

Nickel et al. 1998 ; Pepperkok et al. 2000 ), whereas uptake of the cycling membrane
proteins such as p23, p24, or KDELr is independent of GTP hydrolysis (Malsam
et al. 1999 ; Nickel et al. 1998 ). The mechanism underlying this process is currently
not known [reviewed in Antonny ( 2011 ), Popoff et al. ( 2011a )].
9.3.3 Scission of COPI Vesicles
Arf1 and coatomer are the cytosolic components essential for in vitro formation of
COPI vesicles from Golgi-enriched membranes (Orci et al. 1993 ), chemically
defined large unilamellar vesicles (LUVs) (Bremser et al. 1999 ; Spang
et al. 1998 ), or giant unilamellar vesicles (GUVs) (Faini et al. 2012 ). Consistent
with a role of p24 proteins as membrane machinery, COPI vesicle formation from
LUVs is stimulated by the cytoplasmic peptide of p23 linked to lipid (Bremser
et al. 1999 ). Taken together, the ability to deform the membrane into a COPI bud
and to promote COPI vesicle scission depends exclusively on these two cytosolic
proteins.
Activated Arf1 alone is capable to induce liposome deformation by shallow
insertion of its N-terminal amphipatic helix (Beck et al. 2008 , 2011 ; Krauss
et al. 2008 ; Lundmark et al. 2008 ), i.e. the small GTPase exerts membrane curva-
ture potentiating activity (Faini et al. 2013 ). Further scrutiny indicated that Arf1,
but not a point mutant Arf1 Y35A, forms GTP-dependent dimers, when recruited to
Golgi-enriched membranes. Unlike Arf1 wild type, the variant Arf1 Y35A does not
exhibit membrane curvature potentiating activity and is unable to promote COPI
vesicle formation in vitro (Beck et al. 2008 ). On the other hand, this variant is fully
functional in the recruitment of coatomer, resulting in coated liposomes curved into
buds, arrested in a state before membrane scission. Thus, the ability to form a
COPI-coated bud depends on the recruitment and polymerisation of coatomer and
is independent on the membrane curvature potentiating activity of the small
GTPase. On the other hand this membrane curvature potentiating activity is pre-
requisite for COPI vesicle scission (Beck et al. 2011 ).
COPI vesicles were originally identified under conditions where GTP hydrolysis
was blocked (Barlowe et al. 1994 ; Malhotra et al. 1989 ). Subsequently, in a variety
of studies GTP hydrolysis was either described to be necessary or not needed for the
formation of these vesicles. This controversy has most recently been addressed in
an in vitro reconstitution assay based on semi-intact cell. In this study formation of
COPI vesicles was confirmed to be independent of GTP hydrolysis (Adolf
et al. 2013 ).