The phosducin-like protein PhLP1 is essential for G{beta}{gamma} dimer formation in Dictyostelium discoideum.
Phosducin proteins are known to inhibit G protein-mediated signaling by sequestering Gbetagamma 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 Gbeta (GFP-Gbeta) and GFP-Ggamma 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-Ggamma occurs in wild-type cells but not in phlp1(-) and gbeta(-) cells. Moreover, Gbetagamma 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-Ggamma proteins are present in a complex in wild-type cells, they are free in phlp1(-) and gbeta(-) cells. Collectively, our data strongly suggest the absence of Gbetagamma dimer formation in Dictyostelium cells lacking PhLP1. We propose that PhLP1 serves as a cochaperone assisting the assembly of Gbeta and Ggamma into a functional Gbetagamma complex. Thus, phosducin family proteins may fulfill hitherto unsuspected biosynthetic functions.
Knol
Jaco C
JC
Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
Engel
Ruchira
R
Blaauw
Mieke
M
Visser
Antonie J W G
AJ
van Haastert
Peter J M
PJ
D000818:Animals; D002462:Cell Membrane / Q000737:chemistry; D003600:Cytosol / Q000737:chemistry; D004023:Dictyostelium / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D044387:GTP-Binding Protein beta Subunits / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D044388:GTP-Binding Protein gamma Subunits / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D047468:Immunoprecipitation; D018832:Molecular Chaperones / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000737:chemistry; D021122:Protein Subunits / Q000032:analysis / Q000235:genetics / Q000378:metabolism
C118981:G-protein Beta gamma; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D018832:Molecular Chaperones; D009419:Nerve Tissue Proteins; D021122:Protein Subunits; D049452:Green Fluorescent Proteins; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40