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 166

domain in the cytohesin proteins, is responsible for mediating relief of autoinhibition
and the positive feedback effect (Richardson et al. 2012 ). For Sec7, the HDS1-Arf1-
GTP interaction is required to maintain Golgi localization of Sec7p (Richardson
et al. 2012 ). Interestingly, reduction of Arf1 levels in yeast cells is particularly
detrimental to trans -Golgi function (Gall et al. 2000 ), a result that could potentially
be explained by the need for Sec7 to have a high level of Arf1 for its autocatalytic
activity. One consequence of this mechanism could be to maintain directionality in
trafficking through the Golgi. As described above, the GBF/Gea family of GEFs act
early in the secretory pathway, at the cis -Golgi, and Sec7/BIG GEFs act later at the
trans -Golgi. Interestingly, the HDS1 domain of GBF1, also immediately downstream
of the Sec7 domain, is a direct lipid binding domain that does not require Arf1-GTP
for membrane binding like Sec7p does (Bouvet et al. 2013 ). These results taken
together suggest that one mechanism to drive trafficking forward through the Golgi is
initial recruitment of GBF1/Gea GEFs to produce Arf1-GTP, which then can recruit
the later-acting Sec7/BIG GEFs to Golgi membranes.
Whether Arf6, Arf1, or both are the primary substrates for the cytohesin GEFs has
been a long-standing controversy and is still an open question. However, Arf6-GTP is
more efficient at relieving autoinhibition of cytohesins than Arf1-GTP, both in vitro
and in cells (Cohen et al. 2007 ; DiNitto et al. 2007 ). This fact, combined with the
requirement for acidic phospholipids in cytohesin membrane binding, would restrict
activation of cytohesins to PM or endosomal membranes. These results illustrate the
complexity of Arno activation and shed light on this long-standing debate over the
physiological substrate of cytohesins in cells. The fact that Arf6-GTP can activate
cytoshesin/Arno, that the capacity to activate this GEF is highly dependent on relative
levels of cytohesin/Arno and effectors, and that both Arf6 and Arf1 positive feedback
loops exist, all need to be taken into consideration in evaluating in vivo results. Arf1
is required for specific processes at the PM such as recruitment of proteins to focal
adhesions and in phagocytosis, and Arf1-GTP localizes to these sites (Beemiller
et al. 2006 ;Furmanetal. 2002 ; Kruljac-Letunic et al. 2003 ;Normanetal. 1998 ). In
the forming phagocytic cup, Arf6-GTP is recruited early, followed by Arf1-GTP, at a
stage that requires rapid insertion of new membrane. These results support the idea
that the Arf6-cytohesin-Arf1 cascade may play an important role in processes that
require a high level of Arf protein. Arf6 is less abundant than Arf1 in cells, and since
both Arf1 and Arf6 can recruit effectors such as PI4P 5 kinase and PLD, processes
requiring an acute activation of such effectors may rely on the more abundant Arf1 to
provide an adequate supply. Another process in which this mechanismmay operate is
in the insulin signalling pathway, where both Arf1 and Arf6 were shown to contribute
to activation of PI4P 5 kinase and PLD by cytohesin-2/Arno (Lim et al. 2010 ).