RESUMO
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.
Assuntos
Dictyostelium/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Chaperonas Moleculares/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Animais , Membrana Celular/química , Citosol/química , Dictyostelium/genética , Dimerização , Subunidades beta da Proteína de Ligação ao GTP/análise , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/análise , Subunidades gama da Proteína de Ligação ao GTP/genética , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imunoprecipitação , Chaperonas Moleculares/análise , Chaperonas Moleculares/genética , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/genética , Octoxinol , Polietilenoglicóis/química , Subunidades Proteicas/análise , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismoRESUMO
Retinal phosducin is known to sequester transducin Gbetagamma, thereby modulating transducin activity. Phosducin is a member of a family of phosducin-like proteins (PhLP) found in eukaryotes. Phylogeny of 33 phosducin-like proteins from metazoa, plants and lower eukaryotes identified three distinct groups named phosducin-I-III. We discovered three phlp genes in Dictyostelium, each encoding a phosducin-like protein of a different group. Disruption of the phlp1 gene strongly impaired G-protein signalling, apparently due to mislocalization of Gbetagamma in phlp1-null cells. GFP-Gbeta and GFP-Ggamma are membrane associated in wild-type cells, but cytosolic in phlp1-null cells. Phlp2 disruption is lethal due to a synchronous collapse of the cells after 16-17 cell divisions. Phlp3 disruptants show no abnormal phenotype. These results establish a role for phosducin-like proteins in facilitating folding, localization or function of proteins, in addition to modulating G-protein signalling.