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Retinal phosducin is known to sequester transducin Gbg, 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 identi®ed 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 Gbg in phlp1-null cells. GFP-Gb and GFP-Gg 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.

The mannitol-specific enzyme II (EII), purified free of phospholipid, exhibits a concentration dependence in its specific activity with P-HPr and mannitol as the donor and acceptor substrates, respectively. This concentration dependence, previously observed only in the case of mannitol----mannitol phosphate exchange reaction, indicates that an oligomeric form of the enzyme is responsible for catalyzing the phosphorylation reaction (P-HPr + mannitol----mannitol-P + HPr) as well as the exchange reaction. Kinetic analysis revealed that the monomeric enzyme has a much lower specific activity than the associated species. The specific activity can be increased by raising the steady-state level of phosphorylation of EII and also by adding phospholipid, demonstrating that phosphorylation and the binding of phospholipid facilitate the association process. Kinetic measurements and fluorescence energy transfer measurements demonstrate a strong preference of EII for phospholipids with specific head group and fatty acid composition.

Purified mannitol-specific enzyme II (EIImtl), in the presence of the detergent Lubrol, catalyzes the phosphorylation of mannitol from P-HPr via a classical ping-pong mechanism involving the participation of a phosphorylated EIImtl intermediate. This intermediate has been demonstrated by using radioactive phosphoenolpyruvate. Upon addition of mannitol, at least 80% of the enzyme-bound phosphoryl groups can be converted to mannitol 1-phosphate. The EIImtl concentration dependence of the exchange reaction indicates that self-association is a prerequisite for catalytic activity. The self-association can be achieved by increasing the EIImtl concentration or at low concentrations of EIImtl by adding HPr or bovine serum albumin. The equilibrium is shifted toward the dissociated form by mannitol 1-phosphate, resulting in a mannitol 1-phosphate induced inhibition. Mannitol does not affect the association state of the enzyme. Both mannitol and mannitol 1-phosphate also act as classical substrate inhibitors. The apparent Ki of each compound, however, is approximately equal to its apparent Km, suggesting that mannitol and mannitol 1-phosphate bind at the same site on EIImtl. Due to strong inhibition provided by mannitol and mannitol 1-phosphate in the exchange reaction, the kinetics of this reaction cannot be used to determine whether the reaction proceeds via a ping-pong or an ordered reaction mechanism.

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Phosducin proteins are known to inhibit G protein-mediated signaling by sequestering G␤␥ 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 G␤ (GFP-G␤) and GFP-G␥ 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␥ occurs in wild-type cells but not in phlp1 ؊ and g␤ ؊ cells. Moreover, G␤␥ 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␥ proteins are present in a complex in wild-type cells, they are free in phlp1 ؊ and g␤ ؊ cells. Collectively, our data strongly suggest the absence of G␤␥ dimer formation in Dictyostelium cells lacking PhLP1. We propose that PhLP1 serves as a cochaperone assisting the assembly of G␤ and G␥ into a functional G␤␥ complex. Thus, phosducin family proteins may fulfill hitherto unsuspected biosynthetic functions.Phosducin family proteins have classically been linked to G protein regulation. Both phosducin (Phd) and the related phosducin-like protein (PhLP) have been shown to bind G␤␥ subunits (18,32,53,57,64,69). In so doing, they are thought to function as a cellular "sink" which sequesters free G␤␥ subunits following their dissociation from receptor-activated G proteins (2,19,33,41,58). As G protein-coupled receptors only couple to G␣␤␥ trimers, the sequestration of G␤␥ attenuates transmembrane signaling. Thus, phosducin family proteins may adapt the cell's sensitivity to extracellular signals.Being interested in factors underlying the adaptation of G protein-mediated chemotactic signaling in Dictyostelium discoideum, we recently identified three Dictyostelium Phd-like protein genes (3). To test whether PhLP1, the Dictyostelium protein that is most similar to mammalian Phd and PhLP, is involved in modulating G protein signaling, we analyzed phlp1 knockout cells. Surprisingly, G protein signaling is completely defective rather than enhanced in phlp1 Ϫ cells, which exhibit a phenotype that is remarkably similar to that of g␤ knockout cells. Fluorescence confocal microscopy experiments with cells expressing green fluorescent protein (GFP)-tagged G␤ (GFP-G␤) or GFP-G␥ fusion proteins indicated that G␤␥ complexes are absent from the plasma membrane of phlp1 Ϫ cells, providing a possible explanation for the abrogation of signal transduction (3). These findings suggested that G␤ and G␥ fail to be assembled into a G␤␥ complex in phlp1 Ϫ cells or that the complex is not properly routed to the plasma membrane in these cells. In this paper, we have further investigated the G␤␥ defect in phlp1 Ϫ cells. We show that steady-state levels of (GFP-tagged) G␤ and G␥ subunits are dramatically reduced and that these proteins are detected in the cytosol when the PhL...

Human choriogonadotropin (hCG) is a highly complex glycoprotein consisting of two non-covalently associated subunits. We aimed for the expression of a single-chain hCG in the soil amoebae Dictyostelium discoideum, a host which, in principle, provides simple genetics in combination with complex protein synthesis. To limit anticipated problems in mRNA translation, the first 30 bases of the coding sequence were altered to conform to the Dictyostelium preferred codon usage. We show that, immunologically, active single-chain hCG is indeed produced by Dictyostelium. Furthermore, this single-chain hCG is able to bind to the human luteinizing hormone/CG receptor and elicit a biological response. Its receptorbinding affinity is comparable to single-chain hCG produced by mammalian cells. We conclude that Dictyostelium is able to express bioactive highly complex heterologous glycoproteins.Keywords : gonadotropin; single chain; Dictyostelium; human choriogonadotropin; follitropin.The placental human choriogonadotropin (hCG) is involved in the maintenance of pregnancy in the early stages after conception and has important therapeutic applications. This gonadotropin belongs to the family of glycoprotein hormones, which also include follitropin, lutropin and thyrotropin. These hormones are heterodimeric proteins of around 30 kDa formed by a non-covalent association of a common A subunit and a hormone-specific β subunit. Both the A and β subunits of hCG contain two Nlinked oligosaccharide side chains that have an important impact on its conformation and biological activity. A unique feature of the hCG β-subunit is the carboxy-terminal peptide (CTP) which bears four serine-linked oligosaccharides. The major role of the glycosylated CTP seems to be the prolongation of the circulatory half-life of hCG [1].The biosynthesis of the glycoprotein hormones is a highly complex process. In the last decade, it has become clear that folding, assembly and secretion of gonadotropins is assisted by a large set of chaperones and folding enzymes, residing in the endoplasmic reticulum and the Golgi apparatus [2]. Since both the A and the β subunits contain a so-called cystine knot, it can be anticipated that protein disulphide isomerase plays a key role in the facilitation of the folding process [3]. In addition, it has been shown that the N-linked oligosaccharide side chains are required for proper folding, disulphide formation and secretion of hCG [4].The gonadotropins have been expressed in Chinese Hamster Ovary (CHO) cells, and their recombinant derivatives have bioCorrespondence to P. D. J.