Gβ1γ2 activates phospholipase A2-dependent Golgi membrane tubule formation.

Marie E Bechler,William J Brown

doi:10.3389/fcell.2014.00004

Marie E Bechler, William J Brown

Open Access

https://doi.org/10.3389/fcell.2014.00004

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Abstract

Heterotrimeric G proteins transduce the ligand binding of transmembrane G protein coupled receptors into a variety of intracellular signaling pathways. Recently, heterotrimeric Gβγ subunit signaling at the Golgi complex has been shown to regulate the formation of vesicular transport carriers that deliver cargo from the Golgi to the plasma membrane. In addition to vesicles, membrane tubules have also been shown to mediate export from the Golgi complex, which requires the activity of cytoplasmic phospholipase A2 (PLA2) enzyme activity. Through the use of an in vitro reconstitution assay with isolated Golgi complexes, we provide evidence that Gβ1γ2 signaling also stimulates Golgi membrane tubule formation. In addition, we show that an inhibitor of Gβγ activation of PLA2 enzymes inhibits in vitro Golgi membrane tubule formation. Additionally, purified Gβγ protein stimulates membrane tubules in the presence of low (sub-threshold) cytosol concentrations. Importantly, this Gβγ stimulation of Golgi membrane tubule formation was inhibited by treatment with the PLA2 antagonist ONO-RS-082. These studies indicate that Gβ1γ2 signaling activates PLA2 enzymes required for Golgi membrane tubule formation, thus establishing a new layer of regulation for this process.

Highlights

The formation of transport carriers from the mammalian Golgi complex requires regulation for precise spatial and temporal trafficking within a cell (Yang et al, 2011)
A preparation of bovine brain cytosol (BBC) stimulates Golgi membrane tubule formation in an in vitro reconstitution assay (Cluett et al, 1993; Banta et al, 1995), which can be inhibited by phospholipase A2 (PLA2) inhibitors and stimulated by the addition of purified PAFAH Ib (Bechler et al, 2010, 2012; Ha et al, 2012)
We provide in vitro evidence that heterotrimeric G protein subunits Gβ1γ2 stimulate Golgi membrane tubule formation, which is dependent on PLA2 activity

Summary

Introduction

The formation of transport carriers from the mammalian Golgi complex requires regulation for precise spatial and temporal trafficking within a cell (Yang et al, 2011). Cargo transport from the Golgi complex involves both membrane bound vesicles and membrane tubules. Cargo exiting the trans Golgi network (TGN), such as ts VSV-G, as well as retrograde cargo from the cis Golgi to the endoplasmic reticulum, has been visualized to travel in long, 60–80 nm diameter membrane-bound tubule carriers (Bechler et al, 2012; Ha et al, 2012; Martinez-Alonso et al, 2013). The outward budding of a nascent vesicle initially requires positive membrane curvature that is necessary for forming membrane tubule carriers. Little is known about the mechanisms that regulate the positive curvature needed to form both vesicles and membrane tubules from the Golgi complex (Bechler et al, 2012; Ha et al, 2012)

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