Arf Proteins and Their Regulators: At the Interface Between Membrane Lipids and the Protein Trafficking Machinery - Ras Superfamily Small G Proteins: Biology and Mechanisms 2 - page 158

phosphatidylinositol 4-phosphate, 5-kinase (PIP5K), an enzyme that phosphory-
lates inositol 4-phosphate (PI4P) at the 5-position to generate phosphatidylinositol
4,5-bisphosphate (PI(4,5)P 2 ) (Honda et al. 1999 ). In cells, it is primarily Arf6 that
colocalizes with PIP5K at the PM to generate PI(4,5)P 2 , which in turn stimulates
PM ruffling (Honda et al. 1999 ). At the Golgi, Arf1 recruits and stimulates the
activity of phosphatidylinositol 4-kinase, forming PI4P, an important membrane
lipid for Golgi function (De Matteis and Godi 2004a ).

8.3 New Insights into Coat Recruitment by Arf Proteins

As described above, Arf1 can recruit the COPI coat to early Golgi membranes, as
well as the heterotetrameric clathrin adaptor complexes (AP-1, AP-3, AP-4) and the
three monomeric adaptors (GGA1, GGA2, and GGA3) to trans -Golgi, TGN, and
endosomal membranes. How a single Arf protein can recruit multiple coats to
different membrane sites in cells is still not fully understood, but one important
contribution to specificity comes from the Arf GEFs. In both mammalian and yeast
cells, GBF1 and Gea1/2, respectively, interact directly with COPI (Deng
et al. 2009 ), and knockdown of GBF1 inhibits COPI recruitment to membranes in
mammalian cells (Deng et al. 2009 ; Ishizaki et al. 2008 ; Manolea et al. 2008 ; Szul
et al. 2007 ). In contrast, knockdown of BIG1 and BIG2 Arf1 GEFs inhibits AP-1
and GGA recruitment to the trans -Golgi (Ishizaki et al. 2008 ; Manolea et al. 2008 ).
In yeast, a single class I Arf is responsible for recruiting COPI, AP-1, and GGA
coats to membranes in cells, and hence mechanisms such as Arf GEF-mediated
specificity are required. In mammalian cells, Arfs 3-5 could contribute an addi-
tional layer of specificity. It has been shown recently that a specific subset of Arf
family members (Arf1, Arf4, and Arf5, but not Arf3 or Arf6) are incorporated into
COPI vesicles reconstituted using cytosol (Popoff et al. 2011 ). A full understanding
of the mechanisms determining the specificity of coat recruitment by Arf family
members and their regulators is an important open question in the field.
Important insights into the recruitment of coats by Arf1 have come from recent
structural studies (Ren et al. 2013 ; Yu et al. 2012 ). The structure of Arf1-GTP
bound to a subcomplex of the COPI coat revealed a binding site on the subunit,

which would position the entire COPI complex on the membrane surface in a
conformation very similar to the membrane-bound AP-2/clathrin complex
(Yu et al. 2012 ). COPI, AP-2/clathrin, and AP-1/clathrin complexes share a
remarkable level of structural similarity, although AP-2 is recruited to membranes
by plasma membrane PI(4,5)P 2 rather than an activated Arf protein (Jackson
et al. 2010 ). All of these coats have two large subunits that are symmetrically
located within the complex, which in the case of COPI are the

ʳ

ʲ and ʳ subunits.

Biochemical studies confirmed a second Arf1-GTP binding site on the subunit of

COPI (Yu et al. 2012 ). The structure of Arf1 in complex with the entire central
trunk region of the AP-1 adaptor complex revealed only one of the two Arf1-GTP
binding sites on AP-1, on the

ʲ

ʲ 1 subunit (Ren et al. 2013 ). This Arf1-AP-1 structure