Dictyostelium is an accessible organism for studies of signaling via chemoattractant receptors. Chemoattractant-mediated signaling events and components are reviewed and presented as a series of connected modules, including excitation, inhibition, G protein-independent responses, early gene expression, inositol lipids, PH domain-containing proteins, cyclic AMP signaling, polarization acquisition, actin polymerization, and cortical myosin. The network incorporates information from biochemical, genetic, and cell biological experiments carried out on living cells. The modules and connections represent current understanding, and future information is expected to modify and build upon this structure.

Heterotrimeric G proteins are covalently modified by lipids. Myristoylation of G-protein alpha subunits and prenylation of gamma subunits are stable modifications. In contrast, palmitoylation of alpha subunits is dynamic and thus has the potential for regulating protein function. Indeed, receptor activation of Gs increases palmitate turnover on the alpha subunit, presumably by stimulating deacylation. The enzymes that catalyze reversible palmitoylation of G-protein alpha subunits have not been characterized. Here we report the identification of a palmitoyl-CoA:protein S-palmitoyltransferase activity that acylates G-protein alpha subunits in vitro. Palmitoyltransferase activity is membrane-associated and requires detergent for solubilization. The preferred G-protein substrate for the enzyme activity is the alpha subunit in the context of the heterotrimer. Both myristoylated and nonmyristoylated G-protein alpha subunits are recognized as substrates. The palmitoyltransferase activity demonstrates a modest preference for palmitoyl-CoA over other fatty acyl-CoA substrates. Palmitoyltransferase activity is high in plasma membrane and present at low or undetectable levels in Golgi, endoplasmic reticulum, and mitochondria of rat liver. The subcellular localization of this enzyme activity is consistent with a role for regulated cycles of acylation and deacylation accompanying activation of G-protein signal transduction pathways.

To establish the biological function of thioacylation (palmitoylation), we have studied the heterotrimeric guanine nucleotide-binding protein (G protein) subunits of the pheromone response pathway of Saccharomyces cerevisiae. The yeast G protein ␥ subunit (Ste18p) is unusual among G ␥ subunits because it is farnesylated at cysteine 107 and has the potential to be thioacylated at cysteine 106. Substitution of either cysteine results in a strong signaling defect. In this study, we found that Ste18p is thioacylated at cysteine 106, which depended on prenylation of cysteine 107. Ste18p was targeted to the plasma membrane even in the absence of prenylation or thioacylation. However, G protein activation released prenylation-or thioacylation-defective Ste18p into the cytoplasm. Hence, lipid modifications of the G ␥ subunit are dispensable for G protein activation by receptor, but they are required to maintain the plasma membrane association of G ␤␥ after receptor-stimulated release from G ␣ . The G protein ␣ subunit (Gpa1p) is tandemly modified at its N terminus with amide-and thioester-linked fatty acids. Here we show that Gpa1p was thioacylated in vivo with a mixture of radioactive myristate and palmitate. Mutation of the thioacylation site in Gpa1p resulted in yeast cells that displayed partial activation of the pathway in the absence of pheromone. Thus, dual lipidation motifs on Gpa1p and Ste18p are required for a fully functional pheromone response pathway. INTRODUCTIONLipid modifications anchor heterotrimeric guanine nucleotide-binding proteins (G proteins) to the inner leaflet of the plasma membrane. G protein ␣ subunits are fatty acylated with amide-linked myristate, thioester-linked palmitate, or both. G protein ␥ subunits are prenylated with either farnesyl or geranylgeranyl moieties through stable thioether linkages. Prenylation of ␥ subunits and myristoylation of ␣ subunits are essential for the function of G proteins. These modifications promote plasma membrane association and facilitate high-affinity protein-protein interactions (reviewed in Wedegaertner et al., 1995). The functional consequences of thioacylation are less well understood. Thioacylation does not appear to be a major determinant of membrane avidity, at least in the presence of G ␤␥ subunits, but may play a role in targeting G ␣ specifically to the plasma membrane (Dunphy et al., 1996;Morales et al., 1998;Fishburn et al., 1999;Huang et al., 1999). In vitro studies have demonstrated the importance of thioester-linked lipid in mediating protein-protein interactions of G ␣ subunits. Thioacylation increases the affinity of G s␣ for G ␤␥ approximately fivefold (Iiri et al., 1996) and negatively regulates the interaction between regulators of G protein signaling and G ␣ subunits (Tu et al., 1997). Thus, thioacylation may impact protein-protein interactions, as well as the subcellular distribution of modified proteins.We have investigated thioacylation of G proteins in the genetically tractable organism Saccharomyces cerevisiae. The pheromone res...

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