Shuttling of G Protein Subunits between the Plasma Membrane and Intracellular Membranes

Mariangela Chisari,Deepak Kumar Saini,Vani Kalyanaraman,Narasimhan Gautam

doi:10.1074/jbc.m704246200

Mariangela Chisari, Deepak Kumar Saini + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m704246200

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Abstract

Heterotrimeric G proteins (alphabetagamma) mediate the majority of signaling pathways in mammalian cells. It is long held that G protein function is localized to the plasma membrane. Here we examined the spatiotemporal dynamics of G protein localization using fluorescence recovery after photobleaching, fluorescence loss in photobleaching, and a photoswitchable fluorescent protein, Dronpa. Unexpectedly, G protein subunits shuttle rapidly (t1/2 < 1 min) between the plasma membrane and intracellular membranes. We show that consistent with such shuttling, G proteins constitutively reside in endomembranes. Furthermore, we show that shuttling is inhibited by 2-bromopalmitate. Thus, contrary to present thought, G proteins do not reside permanently on the plasma membrane but are constantly testing the cytoplasmic surfaces of the plasma membrane and endomembranes to maintain G protein pools in intracellular membranes to establish direct communication between receptors and endomembranes.

Highlights

We used a variety of imaging methods on live cells with fluorescent protein-tagged G protein subunits including photoswitchable Dronpa [12] to observe G protein movement
The results show that G protein subunits shuttle rapidly between the PM and endomembranes in cells in the basal state maintaining a pool of G proteins in the endomembranes
G Protein Subunits Shuttle between PM and Endomembranes— To explore the spatiotemporal dynamics of G protein subunit localization in a live cell, we expressed the ␣o subunit tagged with either GFP or Dronpa, a photoswitchable fluorescent protein [12], in CHO cells

Summary

Introduction

We used a variety of imaging methods on live cells with fluorescent protein-tagged G protein subunits including photoswitchable Dronpa [12] to observe G protein movement. A fluorescence resonance energy transfer (FRET)-based G protein sensor was used to examine whether G proteins reside constitutively in the endomembranes. The results show that G protein subunits shuttle rapidly between the PM and endomembranes in cells in the basal state maintaining a pool of G proteins in the endomembranes. We show that the shuttling is likely diffusive and not vesicle-mediated. 2-bromopalmitate (2BP), an inhibitor of palmitoylation [13], inhibited shuttling, suggesting that it may be regulated by acylation

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