Related Galpha subunits play opposing roles during Dictyostelium development.

Of the several known Dictyostelium G protein subunits, the Galpha4 and Galpha5 subunits are the most closely related pair based on phylogenetic analysis and expression patterns, but these subunits perform different roles during development. To investigate potential relationships between these subunits with respect to cell differentiation, chimeric organisms composed of strains lacking or overexpressing either subunit were created and examined for developmental morphogenesis and spore production. Chimeras of galpha4 null and galpha5 null strains or Galpha4 and Galpha5 overexpression strains displayed compensatory morphogenesis, implying that the subunits promote complementary developmental processes. However, chimeras composed of galpha4 null and Galpha5 overexpression strains or galpha5 null and Galpha4 overexpression strains displayed distorted tip morphogenesis, suggesting the strains of these chimeras share common developmental deficiencies. Cells lacking the Galpha5 subunit localized to the prespore region of chimeras similar to the pattern observed for cells overexpressing the Galpha4 subunit, and cells overexpressing the Galpha5 subunit displayed localization patterns similar to galpha4 null mutants. A strain overexpressing both subunits displayed a partial suppression of morphology, gene expression, and cell localization phenotypes associated with the overexpression of the individual Galpha subunit genes, suggesting that each Galpha subunits can inhibit signaling mediated by the other subunit. Overexpression of the Galpha5 subunit inhibited chemotaxis and cGMP accumulation in response to folic acid, indicating that the Galpha5 subunit can inhibit early steps in the Galpha4-mediated signal transduction pathway. The contrasting phenotypes of the Galpha mutants suggest the Galpha4 and Galpha5 subunits provide opposing functions in cell differentiation, localization, and chemotactic responses to folic acid.

Activated Galpha subunits can inhibit multiple signal transduction pathways during Dictyostelium development.

Mutations impairing the GTPase activity of G protein Galpha subunits can result in activated Galpha subunits that affect signal transduction and cellular responses and, in some cases, promote tumor formation. An analogous mutation in the Dictyostelium Galpha4 subunit gene (Q200L substitution) was constructed and found to inhibit Galpha4-mediated responses to folic acid, including the accumulation of cyclic nucleotides and chemotactic cell movement. The Galpha4-Q200L subunit also severely inhibited responses to cAMP, including cyclic nucleotide accumulation, cAMP chemotaxis, and cellular aggregation. An analogous mutation in the Galpha2 subunit (Q208L substitution), previously reported to inhibit cAMP responses (K. Okaichi et al., 1992, Mol. Biol. Cell 3, 735-747), was also found to partially inhibit folic acid chemotaxis. Chemotactic responses to folic acid and cAMP and developmental aggregation were also inhibited by a mutant Galpha5 subunit with the analogous alteration (Q199L substitution). All aggregation-defective Galpha mutants were capable of multicellular development after a temporary incubation at 4 degrees C and this development was found to be dependent on wild-type Galpha4 function. This study indicates that mutant Galpha subunits can inhibit signal transduction pathways mediated by other Galpha subunits.

Folic acid stimulation of the Galpha4 G protein-mediated signal transduction pathway inhibits anterior prestalk cell development in Dictyostelium.

In Dictyostelium discoideum, several G proteins are known to mediate the transduction of signals that direct chemotactic movement and regulate developmental morphogenesis. The G protein alpha subunit encoded by the Galpha4 gene has been previously shown to be required for chemotactic responses to folic acid, proper developmental morphogenesis, and spore production. In this study, cells overexpressing the wild type Galpha4 gene, due to high copy gene dosage (Galpha4HC), were found to be defective in the ability to form the anterior prestalk cell region, express prespore- and prestalk-cell specific genes, and undergo spore formation. In chimeric organisms, Galpha4HC prespore cell-specific gene expression and spore production were rescued by the presence of wild-type cells, indicating that prespore cell development in Galpha4HC cells is limited by the absence of an intercellular signal. Transplanted wild-type tips were sufficient to rescue Galpha4HC prespore cell development, suggesting that the rescuing signal originates from the anterior prestalk cells. However, the deficiencies in prestalk-specific gene expression were not rescued in the chimeric organisms. Furthermore, Galpha4HC cells were localized to the prespore region of these chimeric organisms and completely excluded from the anterior prestalk region, suggesting that the Galpha4 subunit functions cell-autonomously to prevent anterior prestalk cell development. The presence of exogenous folic acid during vegetative growth and development delayed anterior prestalk cell development in wild-type but not galpha4 null mutant aggregates, indicating that folic acid can inhibit cell-type-specific differentiation by stimulation of the Galpha4-mediated signal transduction pathway. The results of this study suggest that Galpha4-mediated signals can regulate cell-type-specific differentiation by promoting prespore cell development and inhibiting anterior prestalk-cell development.

Mutations in the Dictyostelium heterotrimeric G protein alpha subunit G alpha5 alter the kinetics of tip morphogenesis.

Tip morphogenesis during the Dictyostelium developmental life cycle is a process by which prestalk cells sort to form the anterior region of the multicellular organism. We show that the temporal regulation of this morphological process is dependent on the copy number of the Dictyostelium G alpha5 gene. Tip formation is delayed in aggregates of g alpha5 null mutant cells and accelerated in aggregates overexpressing the G alpha5 gene compared to tip formation in wild-type cells. The onset of cell-type-specific gene expression associated with mound formation and tip morphogenesis is also temporally altered in G alpha5 mutants. Tip morphogenesis in chimeric organisms of G alpha5 mutants and wild-type cells is dependent on the copy number of the G alpha5 gene, indicating that G alpha5 function plays an integral role in the intercellular signaling of this stage of development. The G alpha5 gene encodes a G alpha subunit that has 51% identity to the Dictyostelium G alpha4 subunit. Like the G alpha4 gene, the G alpha5 gene is expressed in a subset of cells distributed throughout the multicellular organism, with a distribution that is similar to the anterior-like cell population. Amino acid substitutions in the G alpha5 subunit analogous to substitutions altering guanine nucleotide binding and hydrolysis in other G alpha subunits had no apparent effect on the rate of tip formation when a single copy of the mutant gene was used to replace the wild-type gene. Overexpression of these mutant G alpha5 genes by increased gene dosage resulted in cell death, suggesting that high levels of the altered subunits have detrimental effects during vegetative growth.

The G alpha subunit G alpha 4 couples to pterin receptors and identifies a signaling pathway that is essential for multicellular development in Dictyostelium.

In this paper, we show that the G alpha subunit G alpha 4 couples to pterin receptors and identifies a signalling pathway that is essential for multicellular development in Dictyostelium. G alpha 4 is developmentally regulated, is essential for proper morphogenesis and spore production, and functions cell nonautonomously. We show that G alpha 4 is coupled to receptors (alpha FAR) that activate chemotaxis and adenylyl and guanylyl cyclases in response to folate during the early stages of development and to a late class of folate receptors (beta FAR) that have different specificities for pterins. G alpha 4 is preferentially expressed in cells randomly distributed within the aggregate that are a component of the anterior-like cell population, and it is not detectably expressed in prespore cells. Our results suggest that an endogenous factor, possibly a pterin, produced during multicellular development is a requisite signal for multicellular development, acting through G alpha 4. We propose that the G alpha 4-expressing cells function as a regulatory cell type controlling prespore cell fate, possibly in response to an endogenous pterin. Our results indicate that G alpha 4 and G alpha 2 have parallel functions in mediating cellular responses to folate (pterins) and cAMP, respectively.

Analysis of G alpha 4, a G-protein subunit required for multicellular development in Dictyostelium.

The Dictyostelium G alpha 4 gene encodes a G-protein alpha subunit that is primarily expressed during the multicellular stages of development. g alpha 4 null mutants, created by gene disruption, show aberrant morphological differentiation, reduced levels of prespore gene expression, and a loss of the ability to produce spores. These developmental phenotypes can be rescued by complementation with the wild-type gene. Cells that overexpress the G alpha 4 gene (G alpha 4HC) also show reduced spore production but display an aberrant morphological phenotype distinct from that of g alpha 4 cells. The g alpha 4 phenotype can be partially rescued by the presence of wild-type or G alpha 4HC cells in chimeric organisms, suggesting that G alpha 4-expressing cells produce an intercellular signal that is essential for multicellular development.

Identification of Dictyostelium G alpha genes expressed during multicellular development.

Guanine nucleotide-binding protein (G protein)-mediated signal transduction constitutes a common mechanism by which cells receive and respond to a diverse set of environmental signals. Many of the signals involved in the developmental life cycle of the slime mold Dictyostelium have been postulated to be transduced by such pathways and, in some cases, these pathways have been demonstrated to be dependent on specific G proteins. Using the polymerase chain reaction, we have identified two additional Dictyostelium G alpha genes, G alpha 4 and G alpha 5, that are developmentally regulated. Transcripts from both of these genes are primarily expressed during the multicellular stages of development, suggesting possible roles in cell differentiation or morphogenesis. The entire G alpha 4 gene was sequenced and found to encode a protein consisting of 345 amino acids. The G alpha 4 subunit is homologous to other previously identified G alpha subunits, including the Dictyostelium G alpha 1 (43% identity) and G alpha 2 (41% identity) subunits. However, the G alpha 4 subunit contains some unusual sequence divergences in residues highly conserved among most eukaryotic G alpha subunits, suggesting that G alpha 4 may be a member of another class of G alpha subunits.

Molecular genetic analysis of two G alpha protein subunits in Dictyostelium.

In Dictyostelium, chemotaxis to folate during growth and cAMP during aggregation is controlled via cell surface receptors. To study the role of two G alpha proteins (G alpha 1 and G alpha 2) in these responses, we examined the physiological and biochemical effects of null mutations caused by antisense mutagenesis and gene disruptions. Disruption of G alpha 2 results in an aggregation-deficient phenotype and a loss of cAMP receptor-mediated functions, including activation of adenylate cyclase, guanylate cyclase, and gene expression and in a loss of GTP-mediated decrease in receptor affinity for cAMP, but it has no effect on chemotaxis to folate or folate activation of guanylate cyclase. These phenotypes can be rescued by a vector expressing G alpha 2, suggesting G alpha 2 is coupled to a cAMP receptor but not to folate receptors. Loss of G alpha 1 expression resulted in no visible growth or developmental phenotype, including cAMP- and folate-stimulated responses, suggesting G alpha 1 function is either not essential under standard laboratory conditions or is encoded by multiple genes. Availability of null mutations provides suitable genetic backgrounds for expressing mutant G alpha protein subunits which can then be used to examine the physiological roles of G alpha 1 and G alpha 2. Construction of these gene disruptions was facilitated by using the auxotrophic marker THY1, which allowed for selection of single-copy insertions into the genome.

A family of cyclin homologs that control the G1 phase in yeast.

Two Saccharomyces cerevisiae genes were isolated based upon their dosage-dependent rescue of a temperature-sensitive mutation of the gene CDC28, which encodes a protein kinase involved in control of cell division. CLN1 and CLN2 encode closely related proteins that also share homology with cyclins. Cyclins, characterized by a dramatic periodicity of abundance through the cell cycle, are thought to be involved in mitotic induction in animal cells. A dominant mutation in the CLN2 gene, CLN2-1, advances the G1- to S-phase transition in cycling cells and impairs the ability of cells to arrest in G1 phase in response to external signals, suggesting that the encoded protein is involved in G1 control of the cell cycle in Saccharomyces.

The Saccharomyces cerevisiae CKS1 gene, a homolog of the Schizosaccharomyces pombe suc1+ gene, encodes a subunit of the Cdc28 protein kinase complex.

The Saccharomyces cerevisiae gene CDC28 encodes a protein kinase required for cell cycle initiation. In an attempt to identify genes encoding proteins that interact with the Cdc28 protein kinase, high-copy plasmid suppressors of a temperature-sensitive cdc28 mutation were isolated. One such suppressor, CKS1, was found to encode an 18-kilodalton protein that shared a high degree of homology with the suc1+ protein (p13) of Schizosaccharomyces pombe (67% amino acid sequence identity). Disruption of the chromosomal CKS1 gene conferred a G1 arrest phenotype similar to that of cdc28 mutants. The presence of the 18-kilodalton Cks1 protein in yeast lysates was demonstrated by using Cks-1 specific antiserum. Furthermore, the Cks1 protein was shown to be physically associated with active forms of the Cdc28 protein kinase. These data suggest that Cks1 is an essential component of the Cdc28 protein kinase complex.

Analysis of the Cdc28 protein kinase complex by dosage suppression.

In the interest of identifying components of the Cdc28 protein kinase complex, dosage suppression analysis was performed on temperature-sensitive and dominant negative CDC28 mutations. Dosage suppression is based on a rationale in which elevated expression of wild-type genes can rescue mutations in a target gene as a result of interaction between the respective encoded proteins. Three sequences capable of rescuing a temperature sensitive cdc28 mutation were isolated from a library of wild-type genomic DNA segments in the high copy vector YEp13. Two of these, named CLN1 and CLN2 were found to encode closely related proteins with homology to cyclins. The third, CKS1, encodes an 18K (K = 10(3) Mr) protein that has been shown to be a component of the Cdc28 protein kinase complex and is a homolog of the suc1+ product of fission yeast. A number of dosage suppressors of the CDC28-dn1 dominant negative mutation have been isolated. The one analyzed to date encodes a truncated subunit of the mitochondrial enzyme succinyl-CoA synthetase. The basis for suppression in this case remains to be elucidated.

Invariant phosphorylation of the Saccharomyces cerevisiae Cdc28 protein kinase.

The phosphorylation level of the Saccharomyces cerevisiae Cdc28 protein remained invariant under conditions that resulted in cell cycle arrest in the G1 phase and loss of Cdc28-specific protein kinase activity when the activity was assayed in vitro. These results are in contrast to the proposed regulation of the homologous Cdc2 protein kinase of Schizosaccharomyces pombe.

Protein kinase activity associated with the product of the yeast cell division cycle gene CDC28.

Antibodies raised against the protein encoded by a lacZ-CDC28 in-frame fusion were shown to immunoprecipitate the CDC28 product from yeast cell lysates. The polypeptide p36CDC28 is a phosphoprotein of apparent Mr 36,000. Immune complexes prepared from yeast cell lysates by using anti-CDC28 antibody were found to possess a protein kinase activity, as determined by the transfer of label from [gamma-32P]ATP to a coprecipitated Mr 40,000 protein of unknown identity or function (p40). This activity was absent or thermolabile when extracts were prepared from several different cdc28 temperature-sensitive strains. The protein kinase activity was dependent on Zn2+ and transferred phosphate specifically to serine and threonine residues.