Small G Proteins: Arf Family GTPases in Vesicular Transport - Ras Superfamily Small G Proteins: Biology and Mechanisms 2 - page 185

On the membrane, the GEF interacts with Arf via a highly conserved Sec7
domain (Cherfils et al. 1998 ; Mossessova et al. 1998 ). Insertion of a conserved
glutamate residue (also referred to as 'glutamic finger') in this domain (exposed at
the tip of a hydrophilic loop between helices 6 and 7) into the nucleotide binding
pocket of Arf triggers displacement of GDP by electrostatic competition with the
ʲ

-phosphate of the nucleotide (Beraud-Dufour et al. 1998 ; Renault et al. 2003 ).
Nucleotide exchange causes a conformational change of the switch I and switch II
domains of Arf, affecting the relative position of the interswitch domain. In its
GDP-bound form the myristoylated, amphipathic N-terminal helix of Arf is buried
in a hydrophobic pocket. GTP binding causes a 7-8

shift of the interswitch
region, which concomitantly displaces the N-terminal helix from its pocket (Amor
et al. 1994 ; Goldberg 1998 ). As a result, the myristoylated, amphipathic N-terminal
helix is shallowly inserted into the lipid bilayer and anchors the GTPase to the
membrane (Antonny et al. 1997 ; Franco et al. 1995 , 1996 ). In the prevailing view
the amphipathic N-terminal helix of Arf is oriented parallel to the lipid bilayer when
bound to membranes (Antonny et al. 1997 ; Davies et al. 2003 ), whereas the
myristoyl-anchor is inserted perpendicular into the lipid bilayer (Harroun
et al. 2005 ). In a more recent NMR study it was reported that the myristoyl-
anchor is oriented horizontal to the membrane and partially folds back onto the
N-terminal helix of Arf1 (Liu et al. 2010 ). Due to the use of highly curved bicelles
to mimic natural membranes an altered orientation of membrane-bound Arf cannot
be excluded.
In contrast to the stepwise mode of recruitment of two coat layers in CCVs and
COPII vesicles, activated, membrane-bound Arf recruits the heptameric coat com-
plex coatomer en bloc, (Donaldson et al. 1992 ; Hara-Kuge et al. 1994 ; Palmer
et al. 1993 ). Interestingly, GBF1 seems to interact also directly with ʳ -COP,
providing a molecular explanation how specificity of COPI coat recruitment by
GBF is achieved (Deng et al. 2009 ).
Several interactions between activated Arf1 and coatomer have been described.
Site-directed photo-cross-linking experiments revealed a specific interaction of
Arf1 with the trunk domains of

Å

ʲ 0 -COP and ʳ -COP and ʲ -COP, as well as with

the longin domain of -COP (Sun et al. 2007 ; Zhao et al. 1997 , 1999 ).

More recently the crystal structure of Arf1 bound to a

ʴ

-COP subcomplex was
solved. Structure-guided biochemical analysis of Arf1 binding to a

ʳ / ʶ

ʲ / ʴ -COP

subcomplex revealed common Arf1 binding sites in the ʳ -COP and ʲ -COP subunit.

Furthermore the -COP binding sites are related to the PtdIns(4,5)P 2

binding site in AP2, indicating a similar mechanism of membrane recruitment for
coatomer and APs (Yu et al. 2012 ). This view is further corroborated by a crystal
structure of Arf1 in complex with AP1 (Ren et al. 2013 ). Consistent with multiple
Arf-coatomer interfaces, the ratio of the small GTPase to coatomer was determined
to 3-4 Arf per coatomer (Beck et al. 2009 ; Serafini et al. 1991a ). Binding of
coatomer to the cytoplasmically exposed tail of p24 proteins is believed to further
stabilise the coat on membranes. Binding of dimeric, cytoplasmic tails of p23 and
p24 induces a conformational change within the

ʳ -COP and ʲ

ʳ

-COP subunit, which is