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Molecular and Cellular Biology, September 2005, p. 8393-8400, Vol. 25, No. 18
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.18.8393-8400.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

The Phosducin-Like Protein PhLP1 Is Essential for Gß{gamma} Dimer Formation in Dictyostelium discoideum

Jaco C. Knol,1,{dagger} Ruchira Engel,2,{dagger} Mieke Blaauw,1 Antonie J. W. G. Visser,2 and Peter J. M. van Haastert1*

Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands,1 MicroSpectroscopy Centre, Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands2

Received 31 January 2005/ Returned for modification 18 March 2005/ Accepted 25 May 2005

Phosducin proteins are known to inhibit G protein-mediated signaling by sequestering Gß{gamma} 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 Gß (GFP-Gß) and GFP-G{gamma} 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-G{gamma} occurs in wild-type cells but not in phlp1– and gß– cells. Moreover, Gß{gamma} 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-G{gamma} proteins are present in a complex in wild-type cells, they are free in phlp1– and gß– cells. Collectively, our data strongly suggest the absence of Gß{gamma} dimer formation in Dictyostelium cells lacking PhLP1. We propose that PhLP1 serves as a cochaperone assisting the assembly of Gß and G{gamma} into a functional Gß{gamma} complex. Thus, phosducin family proteins may fulfill hitherto unsuspected biosynthetic functions.

* Corresponding author. Mailing address: Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands. Phone: 31-50-363 4172. Fax: 31-50-363 4165. E-mail: P.J.M.van.Haastert{at}rug.nl.

{dagger} These authors contributed equally to this work.

Molecular and Cellular Biology, September 2005, p. 8393-8400, Vol. 25, No. 18
0022-538X/05/$08.00+0     doi:10.1128/MCB.25.18.8393-8400.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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