The Phosducin-Like Protein PhLP1 Is Essential for Gβγ Dimer Formation in Dictyostelium discoideum

Abstract

Phosducin proteins are known to inhibit G protein-mediated signaling by sequestering Gβγ subunits. However, Dictyostelium discoideum cells lacking the phosducin-like protein PhLP1 display defective rather than enhanced G protein signaling. Here we show that green fluorescent protein (GFP)-tagged Gβ (GFP-Gβ) and GFP-Gγ subunits exhibit drastically reduced steady-state levels and are absent from the plasma membrane in phlp1⁻ cells. Triton X-114 partitioning suggests that lipid attachment to GFP-Gγ occurs in wild-type cells but not in phlp1⁻ and gβ⁻ cells. Moreover, Gβγ dimers could not be detected in vitro in coimmunoprecipitation assays with phlp1⁻ cell lysates. Accordingly, in vivo diffusion measurements using fluorescence correlation spectroscopy showed that while GFP-Gγ proteins are present in a complex in wild-type cells, they are free in phlp1⁻ and gβ⁻ cells. Collectively, our data strongly suggest the absence of Gβγ dimer formation in Dictyostelium cells lacking PhLP1. We propose that PhLP1 serves as a cochaperone assisting the assembly of Gβ and Gγ into a functional Gβγ complex. Thus, phosducin family proteins may fulfill hitherto unsuspected biosynthetic functions.

ACKNOWLEDGMENTS

We thank Peter Devreotes for providing us with the gβ⁻ cell line LW6 and Mark Hink for useful discussions on the FCS data.

The work by R.E. was financially supported by the Research Council for Earth and Life Sciences of the Netherlands Organization for Scientific Research.