Small G Proteins: Arf Family GTPases in Vesicular Transport - Ras Superfamily Small G Proteins: Biology and Mechanisms 2 - page 184

to accommodate cargo of various sizes. Like for the inner layer Sec23/24, isoforms
exist of the outer layer protein Sec31 (Shugrue et al. 1999 ; Stankewich et al. 2006 ;
Tang et al. 2000 ).
9.3 Molecular Mechanism of COPI Vesicle Biogenesis
9.3.1 Coat Recruitment
The initial step in the formation of COPI vesicles is recruitment and activation of
the small GTPase Arf1 from the cytosol to the correct donor membrane. In vitro
cross-linking experiments revealed a specific interaction of a carboxy-terminal site
of Arf1-GDP with dimeric cytoplasmic tails of the type-I transmembrane proteins
p23 and p24 (Gommel et al. 2001 ), suggesting a role of p24 proteins as an Arf-GDP
membrane receptor. This interaction was subsequently confirmed by FRET mea-
surements in living cells (Majoul et al. 2001 ) and pull-down experiments (Contreras
et al. 2004 ).
In addition, recruitment of Arf1 to the cis -Golgi apparatus was reported to
involve the Golgi SNARE membrin/GS27. Mutation of an MxxE motif within
Arf1 impairs recruitment of the small GTPase to the cis -Golgi, whereas recruitment
to the trans -Golgi apparatus is unaffected (Honda et al. 2005 ). Thus additional
signals are likely to exist to ensure specific Arf-GDP recruitment to the trans side of
the Golgi apparatus.
Several lines of evidence indicate that GBF1 in mammals and its homologues
Gea1p and Gea2p in yeast are the GEFs involved in Arf activation. GBF1 is
primarily localised at the ERGIC and the cis -Golgi apparatus, where it shows a
substantial colocalisation with the COPI coat (Claude et al. 1999 ; Garcia-Mata and
Sztul 2003 ; Zhao et al. 2002 ). Overexpression of the catalytically inactive variant
GBF1 E794K (Garcia-Mata et al. 2003 ), injection of antibodies directed against
GBF1 (Zhao et al. 2006 ), or siRNA knock-down of GBF1 (Manolea et al. 2008 ;
Szul et al. 2007 ) causes dissociation of COPI from ERGIC/Golgi membranes. The
exact mechanism of recruitment of GBF1 to membranes of the early secretory
pathway is not completely understood, but seems to involve the small GTPase
Rab1b. Overexpression of a dominant negative mutant of Rab1 causes COPI
dissociation from membranes, an effect which can be reversed by overexpression
of Arf1 or GBF1 (Alvarez et al. 2003 ). Furthermore knock-down experiments of
Rab1b suggest that the small GTPase is required for GBF1 membrane association,
and a direct interaction was mapped to a N-terminal fragment (aa 1-380) of the GEF
(Monetta et al. 2007 ). PtdIns(4)P was also reported to be required for recruitment of
GBF1, and it was proposed that local concentration of PtdIns(4)P might be regu-
lated by Rab1-dependent activation of phosphatidylinositol 4-kinase (PtdIns(4)
KIIIalpha) (Dumaresq-Doiron et al. 2010 ).