G Protein Trafficking - GPCR Signalling Complexes: Synthesis, Assembly, Trafficking and Specificity - page 196

may not substantially affect overall Ga /G b g stoichiometry and thus may not reveal
localization changes. However, model organisms or systems with fewer Ga and
Gb g have proved useful here. Yeast S. cerevisiae genetically lacking the Ga Gpa-1,
and C. elegans embryos depleted of two critical Ga, GOA-1 and GPR-16, using
RNAi, show a redistribution of Gb g from the PM to intracellular sites (Song et al.
1996 ; Gotta and Ahringer 2001 ) . Conversely, in Drosophila photoreceptor cells a
loss of the eye-specific Gb subunits results in a shift of Ga q , the key Ga in photore-
ceptor signaling, from membrane to cytosolic fractions (Kosloff et al. 2003 ; Elia
et al. 2005 ). Taken together, a variety of studies indicate that formation of the het-
erotrimer is an important step in stable localization of both Ga and Gb g at the PM.
A logical hypothesis to explain the above described reciprocal requirement is
that the G protein heterotrimer is formed at an intracellular site before or enroute to
trafficking of the heterotrimer to the PM (Marrari et al. 2007 ) . Current evidence
supports a model in which heterotrimer formation occurs at the cytoplasmic surface
of the ER or Golgi. Moreover, evidence exists for trafficking of nascent G proteins
to the PM via either a Golgi-dependent or -independent pathway. Consistent with
assembly of the heterotrimer at the ER, mutant Gb g that fails to bind Ga and wild
type Gb g that is expressed in the absence of Ga localize predominantly at the ER
(Michaelson et al. 2002 ; Takida and Wedegaertner 2003 ) . Because expression and
interaction with Ga is required to shift Gb g from the ER to the PM, the simplest
explanation is that Ga must interact with Gb g at the ER. Moreover, neither treat-
ment of cells with the Golgi disruptor Brefeldin A nor expression of a dominant
negative Sar1, which blocks ER to Golgi transport, prevented expressed Ga and
Gb g from localizing at the PM (Takida and Wedegaertner 2004 ; Fishburn et al.
1999 ; Gonzalo and Linder 1998 ). These studies suggested that not only would the
heterotrimer form before trafficking to the Golgi, i.e. , at the ER, but, in addition,
trafficking to the PM would not require the Golgi. On the other hand, some studies
have argued that Ga and Gb g first interact at the Golgi and/or that the Golgi is
required for movement of G proteins to the plasma membrane. One study observed
apparent Golgi localization of co-expressed Gb g and a palmitoylation site mutant of
Ga i , and thus suggested that heterotrimer interaction and Ga palmitoylation takes
place at the Golgi (Michaelson et al. 2002 ). A recent study showed that DHHC-3
and DHHC-7 are required for palmitoylation of Ga i , G a s and Ga q , and that these
palmitoyl acyltransferases are localized to the Golgi (Tsutsumi et al. 2009 ) .
Moreover, others have argued that the Golgi is the main subcellular site for palmi-
toylation of all peripheral membrane proteins (Rocks et al. 2010 ) . Although the
above studies suggest a functional Golgi may be required for palmitoylation and
trafficking to the PM, they do not address whether or not the heterotrimer would
first form at the ER before moving to the Golgi. Some light was shed on the question
of the involvement of the Golgi in G protein trafficking to the PM by a report show-
ing that the trafficking pathway depends on the protein complexes being formed.
A functional Golgi was not required for PM localization of overexpressed Ga and
Gb g, as shown previously (Takida and Wedegaertner 2004 ); however, when a GPCR
was also overexpressed, the Golgi was indeed required for PM localization of Ga ,
Gb g and GPCR, suggesting that a heterotrimer in a complex with a GPCR takes a