G Protein Trafficking - GPCR Signalling Complexes: Synthesis, Assembly, Trafficking and Specificity - page 205

lated at the Golgi, it is kinetically trapped there through strong hydrophobic anchoring
to the Golgi membrane; and (5) specific transport, probably vesicle mediated, moves
palmitoylated proteins from the Golgi to the PM. Most Ga are palmitoylated, and
thus this model has relevance for defining and understanding the constitutive
movement of G proteins.
Accordingly, recent data has supported rapid G protein shuttling between the PM
and Golgi in a palmitoylation-dependent manner. Studies have used imaging tech-
niques such as fluorescence recovery after photobleaching (FRAP), fluorescence
loss in photobleaching (FLIP) and photoactivation to analyze the movement of
G protein subunits fused to fluorescent proteins. Overexpression of all three sub-
units of a heterotrimer, in this case Ga o , G b 1 and several different Gg, including Gg 2 ,
Gg 3 , G g 9 and Gg 11 , showed substantial localization of the G protein to both the PM
and an intracellular location, likely Golgi (Chisari et al. 2007 ) . However, it should
be noted that a number of studies overexpressing all three G protein subunits have
observed predominant PM localization with little or no intracellular staining.
Nonetheless, the above study used various live cell imaging techniques to demon-
strate that there was a rapid and constitutive movement of all expressed G subunits
between the Golgi and PM in both the anterograde and retrograde direction (Chisari
et al. 2007 ). Based on the identical profile of movement of all three subunits, it
appears that the inactive heterotrimer is constitutively trafficking, rather than Ga
and Gb g moving separately. Interestingly, the PM-Golgi shuttling was blocked by
treatment with the palmitoylation inhibitor 2-bromopalmitate (2-BP) consistent
with the model that a palmitoylation and depalmitoylation cycle mechanistically
underlies this constitutive G protein trafficking. Both the retrograde and anterograde
movement occurred within seconds and inhibitors of vesicle trafficking failed to
block either direction of trafficking, suggesting that constitutive G protein trafficking
occurs via diffusion (Chisari et al. 2007 ; Saini et al. 2009 ) . Another recent study
supported the model that G proteins can constitutively move between the PM and
Golgi (Tsutsumi et al. 2009 ). Photoactivation and FRAP were used to follow the
traf fi cking of G a q when co-expressed with Gb 1 g 2 , and indeed movement between
the PM and Golgi in both anterograde and retrograde directions was observed.
Similar to the studies with the Ga o -containing heterotrimers (Chisari et al. 2007 ) ,
the constitutive shuttling of Ga q -containing heterotrimers was blocked by 2-BP
treatment (Tsutsumi et al. 2009 ) . Moreover, the G a q study implicated DHHC-3 and
DHHC-7 as Golgi-localized palmitoyl acyltransferases responsible for palmitoyla-
tion of Ga q . The movement of Ga q between the PM and Golgi occurred in approxi-
mately 10 min, which was substantially slower than the shuttling of Ga o -containing
heterotrimers which happened in a matter of seconds. The reason for this temporal
difference awaits further experimentation.
Based on the above described studies and on extensive experiments with model
palmitoylated proteins, such as H- and N-Ras (Rocks et al. 2005, 2010 ; Goodwin
et al. 2005 ), a model for constitutive movement of heterotrimeric G proteins has
been described (Saini et al. 2009 ). As with Ras and other palmitoylated peripheral
membrane proteins, the model states that an inactive heterotrimer is constitutively
depalmitoylated at the PM and then moves rapidly by diffusion to the Golgi, where