The Small GTPase RalA Controls Exocytosis of Large Dense Core Secretory Granules by Interacting with ARF6-dependent Phospholipase D1

Nicolas Vitale,Jacques Mawet,Jacques Camonis,Romano Regazzi,Marie-France Bader,Sylvette Chasserot-Golaz

doi:10.1074/jbc.m413748200

Abstract

RalA and RalB constitute a family of highly similar Ras-related GTPases widely distributed in different tissues. Recently, active forms of Ral proteins have been shown to bind to the exocyst complex, implicating them in the regulation of cellular secretion. Since RalA is present on the plasma membrane in neuroendocrine chromaffin and PC12 cells, we investigated the potential role of RalA in calcium-regulated exocytotic secretion. We show here that endogenous RalA is activated during exocytosis. Expression of the constitutively active RalA (G23V) mutant enhances secretagogue-evoked secretion from PC12 cells. Conversely, expression of the constitutively inactive GDP-bound RalA (G26A) or silencing of the RalA gene by RNA interference led to a strong impairment of the exocytotic response. RalA was found to co-localize with phospholipase D1 (PLD1) at the plasma membrane in PC12 cells. We demonstrate that cell stimulation triggers a direct interaction between RalA and ARF6-activated PLD1. Moreover, reduction of endogenous RalA expression level interfered with the activation of PLD1 observed in secretagogue-stimulated cells. Finally, using various RalA mutants selectively impaired in their ability to activate downstream effectors, we show that PLD1 activation is essential for the activation of secretion by GTP-loaded RalA. Together, these results provide evidence that RalA is a positive regulator of calcium-evoked exocytosis of large dense core secretory granules and suggest that stimulation of PLD1 and consequent changes in plasma membrane phospholipid composition is the major function RalA undertakes in calcium-regulated exocytosis.

Highlights

RalA and RalB constitute a family of proteins within the Ras branch of monomeric GTPases [1]
RalA Binds to ARF6-activated phospholipase D1 (PLD1) in Stimulated PC12 Cells—Because Ral proteins are known regulators of ARF-dependent PLD1 [8, 9], a protein that plays a key role in exocytosis in chromaffin and PC12 cells [35, 44], we investigated whether PLD1 might be an effector for RalA in the exocytotic pathway
The exocyst complex comprises eight proteins originally identified in the budding yeast, where they have been shown to be essential for exocytosis [17, 46]

Summary

Introduction

RalA and RalB constitute a family of proteins within the Ras branch of monomeric GTPases [1]. Like most Ras family GTPases, Ral proteins have been implicated in the regulation of various cell biological processes, including oncogenic transformation, endocytosis, and actin-cytoskeleton dynamics [3]. Activated Ral proteins have been implicated in the targeting of Golgi-derived vesicles to the basolateral membrane in epithelial cells [15, 25]. This possibility arose from the discovery that two subunits of the exocyst complex are downstream binding partners of active RalA and RalB (14 –16, 26). By promoting assembly of the exocyst complex [26], Ral proteins have been proposed to regulate exocyst-mediated vesicle delivery to appropriate fusion sites at the plasma membrane

Methods

Results

Conclusion