Loss-of-function mutations identified in the Helical domain of the G protein alpha-subunit, G alpha2, of Dictyostelium discoideum

The guanine nucleotide binding regulatory proteins (G proteins) play essential roles in a wide variety of physiological processes, such as vision, hormone responses, olfaction, immune response, and development. The heterotrimeric G proteins consist of alpha-, beta-, and gamma-subunits and act as molecular switches to relay information from transmembrane receptors to intracellular effectors. The switch mechanism is a function of the inherent GTPase activity of the alpha-subunit. The alpha-subunit is comprised of two domains, the GTPase domain and the Helical domain. The GTPase domain performs all of the known alpha-subunit functions while little is know about the role of the Helical domain. To gain a better understanding of alpha-subunit function, we performed a screen for loss-of-function mutations, using the G alpha2-subunit of Dictyostelium. G alpha2 is essential for the developmental life cycle of Dictyostelium. It is known that the loss of G alpha2 function results in a failure of cells to enter the developmental phase, producing a visibly abnormal phenotype. This allows the easy identification of amino acids essential to G alpha2 function. A library of random point mutations in the g alpha2 cDNA was constructed using low fidelity polymerase chain reaction (PCR). The library was then expressed in a g alpha2 null cell line and screened for loss-of-function mutations. Mutations were identified in isolated clones by sequencing the g alpha2 insert. To date, sixteen single amino acids changes have been identified in G alpha2 which result in loss-of-function. Of particular interest are seven mutations found in the Helical domain of the alpha-subunit. These loss-of-function mutations in the alpha-subunit Helical domain may provide important insight into its function.     

Cloning and targeted mutations of G alpha 7 and G alpha 8, two developmentally regulated G protein alpha-subunit genes in Dictyostelium.

GTP-binding protein (G protein)-mediated signal transduction pathways play essential roles during the aggregation and differentiation process of Dictyostelium. In addition to the five known G protein alpha-subunit genes, we recently identified three novel alpha-subunit genes, G alpha 6, G alpha 7, and G alpha 8, using the polymerase chain reaction technique. We present here a more complete analysis of G alpha 7 and G alpha 8. The cDNAs of these two genes were cloned, and their complete nucleotide sequences were determined. Sequence analyses indicate that G alpha 8 possesses some unusual features. It lacks the "TCATDT" motif, a sequence of amino acids highly conserved among G alpha subunits, and has an additional 50 amino acids at its C-terminus consisting of long stretches of asparagine. Moreover, G alpha 8 is unusually resistant to protease digestion, which may indicate a slow GTP hydrolysis rate. The possible functions of these alpha-subunits were assessed by generating mutants lacking G alpha 7 or G alpha 8 by gene targeting through homologous recombination and by overexpressing G alpha 7 or G alpha 8 protein. Overexpression of G alpha 7 resulted in abnormal morphogenesis starting at the slug stage, whereas analysis of the other strains failed to reveal any obvious growth or developmental defects under either normal or stressful conditions. The implications of these results are discussed.     

Regulation of guanylate cyclase by a guanine nucleotide binding protein, G alpha 2, in Dictyostelium discoideum.

Binding of cyclic AMP (cAMP) to the cell surface receptor induces a transient activation of guanylate cyclase in Dictyostelium discoideum. A frigid mutant (HC85) which lacks G alpha 2, a guanine nucleotide binding protein, does not respond to cAMP. We found that 2,3-dimercapto-1-propanol (BAL) induced a continuous activation both in the frigid and in its parents. Therefore, the BAL-induced continuous activation of guanylate cyclase is independent of G alpha 2. We also found that cAMP enhanced the BAL-induced continuous activation in the frigid mutant. This result suggests that an unidentified signal transduction mechanism from the cAMP-receptor besides the one involving G alpha 2 plays a role in the enhancement of activation. Lastly, we found that the BAL-induced continuous activation was terminated by cAMP in the parental strain, but not in the frigid mutant. Therefore, the cAMP-induced suppression on the BAL-induced continuous activation is mediated through G alpha 2.     

Role of a guanine nucleotide binding protein, G alpha 2, in regulation of adenylate cyclase in Dictyostelium discoideum

Two substances, cAMP and 2,3-dimercapto-1-propanol (BAL) are known to induce transient activation of adenylate cyclase in Dictyostelium discoideum. A frigid mutant (HC85) has a deletion in a gene for G alpha 2, a guanine nucleotide binding protein and cannot activate the cyclase in response to cAMP. We found that BAL induced activation in the frigid mutant. This result suggests that the BAL-induced activation is independent of G alpha 2 and that BAL mimics a role of activated G alpha 2. We also found that cAMP promoted the BAL-induced activation. This result suggests that cAMP plays a role in activation through a mechanism in which G alpha 2 is not involved. We lastly showed that continuous cAMP stimulation could not inhibit the BAL-induced activation in the frigid mutant. Since the cAMP-induced inhibition observed in the wild type strain (NC4) proceeds with the time course identical to the cAMP-induced adaptation (Oyama, submitted), this result suggests that G alpha 2 is involved in adaptation of adenylate cyclase.     

Loss-of-function mutations identified in the Helical domain of the G protein α-subunit,Gα2, of Dictyostelium discoideum

The guanine nucleotide binding regulatory proteins (G proteins) play essential roles in a wide variety of physiological processes, such as vision, hormone responses, olfaction, immune response, and development. The heterotrimeric G proteins consist of α-, β-, and γ-subunits and act as molecular switches to relay information from transmembrane receptors to intracellular effectors. The switch mechanism is a function of the inherent GTPase activity of the α-subunit. The α-subunit is comprised of two domains, the GTPase domain and the Helical domain. The GTPase domain performs all of the known α-subunit functions while little is know about the role of the Helical domain. To gain a better understanding of α-subunit function, we performed a screen for loss-of-function mutations, using the Gα2-subunit of Dictyostelium. Gα2 is essential for the developmental life cycle of Dictyostelium. It is known that the loss of Gα2 function results in a failure of cells to enter the developmental phase, producing a visibly abnormal phenotype. This allows the easy identification of amino acids essential to Gα2 function. A library of random point mutations in the gα2 cDNA was constructed using low fidelity polymerase chain reaction (PCR). The library was then expressed in a gα2 null cell line and screened for loss-of-function mutations. Mutations were identified in isolated clones by sequencing the gα2 insert. To date, sixteen single amino acids changes have been identified in Gα2 which result in loss-of-function. Of particular interest are seven mutations found in the Helical domain of the α-subunit. These loss-of-function mutations in the α-subunit Helical domain may provide important insight into its function.     

Cytochemical localization of alkaline phosphatase in the cell membrane of Dictyostelium discoideum amebae

We demonstrated that alkaline phosphatase was localized on the cell membrane of Dictyostelium discoideum amebae and on isolated plasma membranes. The enzyme activity was specifically inhibited by 0.01 M KCN or cysteine. The same method could also be applied to baker's yeast and MDCK cells (dog kidney cells in vitro).     

The Effect of Divalent Cations on Aggregation of Dictyostelium discoideum

Following the initiation of development, amoebae of Dictyostelium discoideum aggregate chemotactically toward cyclic AMP (cAMP). Adenyl cyclase, cAMP phosphodiesterase, and cAMP binding sites all increase 20–40 fold during the first few hours of development. It has been shown that addition of 1 mM EDTA and 5 mM MgCl₂ accelerates the aggregation process. Likewise, the calcium ionophore, A23187, leads to precocious aggregation while 4 × 10⁻⁵ M progesterone considerably delays it These treatments have now been shown to result in increased accumulation of adenyl cyclase in the case of EDTA and Mg²⁺ or the ionophore and greatly decreased accumulation in the case of the steroid.
Treatment with EDTA and Mg²⁺ or the ionophore has been shown not only to accelerate aggregation in wild-type amoebae but to overcome complete blocks to aggregation in certain mutant strains. We have found that addition of Mn²⁺ will also permit aggregation of mutant cells otherwise unable to aggregate. This divalent ion, unlike EDTA and Mg²⁺ or the ionophore, was shown to directly stimulate adenyl cyclase. Calcium ions were also found to affect the enzyme such that at Ca²⁺ concentrations found within the cells the great majority of the activity is inhibited. Manganese ions can overcome the inhibition by Ca²⁺.
These findings show that conditions which stimulate aggregation result in increased activity of adenyl cyclase either by increased accumulation of the enzyme or by increased activity of the available enzyme, and support the proposed central role of adenyl cyclase in aggregation.     

A RAS-encoded protein in Dictyostelium discoideum is acylated and membrane-associated

Dictyostelium dfscoideum synthesizes a 23000 M_r protein, p23^dd-ras, closely related to the mammalian oncogene-encoded protein p21^ras. To investigate the subcellular localization of P23^dd-ras, conditions were optimized to reduce protein degradation following cell breakage. Subcellular fractionation of D. discoideum showed that p23^dd-ras was associated predominantly with the membrane fraction during both vegetative growth and differentiation, in the absence of suitable protease inhibitors considerable amounts of a truncated form of p23^dd-ras were recovered in the cytosol fraction, suggesting that intact p23^dd-ras is attached to the membrane by a short terminal peptide sequence. Radio-isotope labelilng of D. discoideum with myristic acid or palmitic acid in the presence of excess un-labelled acetate resulted in radio-isotope incorporation into a select group of proteins including p23^dd-ras. No acyl label appeared in the truncated cytoplasmic form of p23^dd-ras when ceil breakage was performed In the absence of suitable protease inhibitors, indicating that the acyl group is associated with the short terminal peptide that is cleaved. These data suggest that p23^dd-ras like its mammailan counterpart, is acylated and associated with the plasma membrane. There was no evidence during a 30-minute pulse of methionine for a cytoplasmic precursor to the membrane-bound p23^dd-ras suggesting that the turnover of the presumptive precursor must be much more rapid in D. discoideum than for pro-p21^ras in mammalian cells.     

Characterization of the extra-large G protein alpha-subunit XLalphas. I. Tissue distribution and subcellular localization

Our group previously described a new type of G protein, the 78-kDa XLalphas (extra large alphas) (Kehlenbach, R. H., Matthey, J., and Huttner, W. B. (1994) Nature 372, 804-809 and (1995) Nature 375, 253). Upon subcellular fractionation, XLalphas labeled by ADP-ribosylation with cholera toxin was previously mainly detected in the bottom fractions of a velocity sucrose gradient that contained trans-Golgi network and was differentially distributed to Galphas, which also peaked in the top fractions containing plasma membrane. Here, we investigate, using a new antibody specific for the XL domain, the tissue distribution and subcellular localization of XLalphas and novel splice variants referred to as XLN1. Upon immunoblotting and immunofluorescence analysis of various adult rat tissues, XLalphas and XLN1 were found to be enriched in neuroendocrine tissues, with a particularly high level of expression in the pituitary. By both immunofluorescence and immunogold electron microscopy, endogenous as well as transfected XLalphas and XLN1 were found to be predominantly associated with the plasma membrane, with only little immunoreactivity on internal, perinuclear membranes. Upon subcellular fractionation, immunoreactive XLalphas behaved similarly to Galphas but was differentially distributed to ADP-ribosylated XLalphas. Moreover, the bottom fractions of the velocity sucrose gradient were found to contain not only trans-Golgi network membranes but also certain subdomains of the plasma membrane, which reconciles the present with the previous observations. To further investigate the molecular basis of the association of XLalphas with the plasma membrane, chimeric proteins consisting of the XL domain or portions thereof fused to green fluorescent protein were analyzed by fluorescence and subcellular fractionation. In both neuroendocrine and non-neuroendocrine cells, a fusion protein containing the entire XL domain, in contrast to one containing only the proline-rich and cysteine-rich regions, was exclusively localized at the plasma membrane. We conclude that the physiological role of XLalphas is at the plasma membrane, where it presumably is involved in signal transduction processes characteristic of neuroendocrine cells.     

A stalk-specific localization of trehalase activity in Dictyostelium discoideum.

By utilizing ultra-microtechniques, trehalase activity was followed in specific cell types during the differentiation cycle of Dictyostelium discoideum. When whole organisms were assayed, trehalase activity was found to be high in the early stages of differentiation, decreased to its lowest point at 14 h, and then increased at the end of the cycle. By microdissection of freeze-dried individuals, the activity of trehalase could be followed during the migration of pre-stalk and pre-spore cells. No activity was observed at any stage of spore cell development, whereas stalk cells showed a rapid increase in activity upon maturation. An increasing gradient of activity was found from the apex of the stalk toward the base. This localization of trehalase in stalk cells resolves some contradictory results in the literature concerning the role of the enzyme during differentiation.     

A sequence of dependent stages in the development of Dictyostelium discoideum.

Eleven marker enzymes which accumulate during discrete stages of development in Dictyostelium discoideum were followed in two independent temperature-sensitive mutant strains. Strain TS2 has a temperature-sensitive period during aggregation and remains as a smooth lawn at the nonpermissive temperature (27°C). It develops normally at 22°C. Strain DTS6 has a temperature-sensitive lesion in the post-aggregation stage and fails to form slugs at 27°C. Early enzymes accumulate in these strains at the nonpermissive temperature but late stage-specific enzymes fail to accumulate at 27°C. The pattern of accumulation of specific enzymes in these and other morphological mutants defines a linear dependent pathway of at least eight steps which determines temporal differentiation in this organism. Development in Dictyostelium is also dependent on environmental cues which determine the onset of differentiation and the preparation for culmination.     

A developmentally regulated membrane protein gene in Dictyostelium discoideum is also induced by heat shock and cold shock.

We have analyzed the expression of the Dictyostelium gene P8A7 which had been isolated as a cDNA clone from an early developmentally regulated gene. The single genomic copy generated two mRNAs which were subject to different control mechanisms: while one mRNA (P8A7S) was regulated like the cell-type-nonspecific late genes, the other one (P8A7L) was induced during development, when cells were allowed to attach to a substrate, and when cells were subjected to stress, such as heat shock and cadmium. Interestingly the same induction was also observed with cold shock. RNA processing was inhibited by heat and cold shock, leading to nuclear accumulation of a precursor. The translated region of the cDNA was common to both mRNAs and encoded an unusually hydrophobic peptide with the characteristics of a membrane protein.     

Regulation and function of G alpha protein subunits in Dictyostelium

We have examined the developmental regulation and function of two G alpha protein subunits, G alpha 1 and G alpha 2, from Dictyostelium. G alpha 1 is expressed in vegetative cells through aggregate stages while G alpha 2 is inducible by cAMP pulses and preferentially expressed in aggregation. Our results suggest that G alpha 2 encodes the G alpha protein subunit associated with the cAMP receptor and mediates all known receptor-activated intracellular signal transduction processes, including chemotaxis and gene regulation. G alpha 1 appears to function in both the cell cycle and development. Overexpression of G alpha 1 results in large, multinucleated cells that develop abnormally. The central role that these G alpha proteins play in signal transduction processes and in controlling Dictyostelium development is discussed.     

Chromatographic behavior of cyclic AMP dependent protein kinase and its subunits from Dictyostelium discoideum

An adenosine cyclic 3',5'-monophosphate (cAMP) dependent protein kinase has recently been shown to exist in Dictyostelium discoideum and to be developmentally regulated. In this report we have followed the chromatographic behavior of both the holoenzyme and its subunits. A cAMP-dependent holoenzyme could be obtained from the 100000 g soluble fraction after passage through DE-52 cellulose (pH 7.5) and Sephacryl S300. Under conditions of low pH the holoenzyme could be further purified by flat-bed electrofocusing (pI = 6.8). Application of the holoenzyme to electrofocusing at high pH resulted in dissociation of the holoenzyme into a cAMP binding component (pI = 6.1) and a cAMP-independent catalytic activity (pI = 7.4). Dissociation of the holoenzyme into subunits also occurred during histone affinity chromatography and gel filtration chromatography (S300) in the presence of a dissociating buffer. Although the subunit structure was clearly evident during chromatography, the holoenzyme could not be dissociated by simple addition of cAMP to the extract. The catalytic subunit could be purified further by CM-Sephadex, DE-52 cellulose (pH 8.5), histone affinity, and hydrophobic chromatography. The regulatory subunit was further purified by DE-52 cellulose (pH 8.5) and cAMP affinity chromatography. Proof that the cAMP binding activity and the cAMP-independent catalytic activity were in fact the regulatory and catalytic subunits was shown by reconstitution of the cAMP-dependent holoenzyme from the purified subunits. By using these separation procedures, one can obtain from extracts of Dictyostelium the subunits that are free of each other as well as free of any endogenous protein substrates.     

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.