Regulation of Constitutive Cargo Transport from the trans-Golgi Network to Plasma Membrane by Golgi-localized G Protein βγ Subunits

Abstract

Observations of Golgi fragmentation upon introduction of G protein ␤␥ (G␤␥) subunits into cells have implicated G␤␥ in a pathway controlling the fission at the trans-Golgi network (TGN) of plasma membrane (PM)-destined transport carriers. However, the subcellular location where G␤␥ acts to provoke Golgi fragmentation is not known. Additionally, a role for G␤␥ in regulating TGN-to-PM transport has not been demonstrated. Here we report that constitutive or inducible targeting of G␤␥ to the Golgi, but not other subcellular locations, causes phospholipase C-and protein kinase D-dependent vesiculation of the Golgi in HeLa cells; Golgi-targeted ␤ 1 ␥ 2 also activates protein kinase D. Moreover, the novel G␤␥ inhibitor, gallein, and the G␤␥-sequestering protein, GRK2ct, reveal that G␤␥ is required for the constitutive PM transport of two model cargo proteins, VSV-G and ss-HRP. Importantly, Golgi-targeted GRK2ct, but not a PM-targeted GRK2ct, also blocks protein transport to the PM. To further support a role for Golgi-localized G␤␥, endogenous G␤ was detected at the Golgi in HeLa cells. These results are the first to establish a role for Golgi-localized G␤␥ in regulating protein transport from the TGN to the cell surface.Heterotrimeric G proteins, composed of ␣, ␤, and ␥ subunits, are involved in a wide variety of signaling responses and act by coupling heptahelical G protein-coupled receptors (GPCRs) 2 to intracellular effector proteins. In its inactive state, the G␣ subunit is bound to GDP and is also tightly bound to the G␤␥ subunits. Upon receptor activation by ligand binding, the GPCR catalyzes the exchange of GDP for GTP on the G␣, resulting in dissociation of G␣ and G␤␥. The separated subunits are then able to elicit their own signaling cascades. Hydrolysis of GTP by G␣, followed by re-association of G␣ and G␤␥, completes the cycle (1, 2).

Inducible Inhibition of Gβγ Reveals Localization-dependent Functions at the Plasma Membrane and Golgi

“…We also use GRK2ct-R587Q (RQ), a ␤␥-binding deficient mutant. WT GRK2ct inhibits ␤␥-mediated transport of VSV-G from the Golgi to the PM, upon temperature release, as demonstrated previously (5). Less than 10% of WT GRK2ct-positive cells display PM localization of VSV-G.…”

“…Basolaterally directed transport from the Golgi to the PM has already been shown to be PKD-dependent, whereas the transport of cargo proteins targeted to apical regions occurs independently of PKD (9). Secretion of insulin in response to glucose was found to require PKD signaling at the Golgi (15), and previous results in our laboratory showed that ␤␥ is the upstream effector of PKD at the TGN for regulating Golgi to PM transport of VSV-G and secreted signal sequence horseradish peroxidase (ssHRP), two model basolaterally targeted cargoes (5). Thus, there is a role for ␤␥ in the regulation of basolateral cargo trafficking, but to date, no link has been made between ␤␥ and distribution of cargo toward distinct membrane domains.…”

“…A C-terminal fragment of GPCR kinase 2 (GRK2ct) binds ␤␥ and has been widely used to inhibit ␤␥ signaling in cells (16). Previous work in our laboratory resulted in targeted inhibition of ␤␥ by fusing GRK2ct to a Golgi-localized mutant KDEL receptor (5). Although this was useful in determining that Golgi-localized ␤␥ was responsible for vesicle fission, it resulted in constitutive inhibition of ␤␥ at the Golgi.…”

“…Overexpression of ␤␥ or other key regulators, such as PKD, of the Golgi-localized signaling pathway discussed above can cause fragmentation of the Golgi (5,6,17). Although such fragmentation is thought to simply represent overactivation of a pathway that regulates transport vesicle formation at the Golgi, it also begs the question of whether endogenous ␤␥ is involved in any processes that stimulate Golgi fragmentation.…”

Microarray profiling of preselected CHO host cell subclones identifies gene expression patterns associated with in‐creased production capacity

“…To our current understanding, the activity and regulation of the trans-Golgi network (TGN) is a highly complex protein sorting machinery. A distinct signalling cascade guides the cargo either to the plasma membrane [53][54][55] or towards the endosome [56,57]. In recent studies, it was successfully shown that this tight regulation of TGN is a valid target for CHO engineering by heterologous over-expression of ceramide transfer protein (CERT), which is a component of protein kinase D (PKD)-mediated transfer processes [58].…”