A G-protein beta-subunit is essential for Dictyostelium development.

Abstract

Recent studies have demonstrated that G-protein-linked signal transduction pathways play a significant role in the developmental program of the simple eukaryotic organism Dictyostelium. We have reported previously the isolation of a G-protein 13-subunit and present here a more complete analysis of this gene. Low-stringency Southern blots and RFLP mapping studies suggest that the 13-subunit is a unique gene found on linkage group If. Its deduced amino acid sequence of 347 residues is -60% identical to those of the human, Drosophila, and Caenorhabditis elegans 13-subunits. The carboxy-terminal 300 residues are about 70% identical; the amino-terminal 50 residues are quite divergent, containing only 10 identities. At all stages of growth and development, a single 1.9-kb 13-subunit mRNA is present at a high level, and a specific antibody detects a single 37-kD protein. We propose that G-protein heterotrimers are formed when this 13-subunit couples with each of the eight distinct G-protein a-subunits that are transiently expressed during development. Targeted disruption of the 13-subunit gene had no effect on the viability of haploid cells, but resulted in the inability of cells to aggregate.

Gγ inDictyostelium: Its Role in Localization of Gβγ to the Membrane Is Required for Chemotaxis in Shallow Gradients

“…Although g␤ Ϫ cells were completely defective in aggregation and most signaling events (Wu et al, 1995), HisG␤/g␤ Ϫ cells developed appropriately into spores and stalks (our unpublished results). The HisG␤ protein was not largely overexpressed, consistent with previous data suggesting tight control of G␤ expression (Lilly et al, 1993). Through purification of HisG␤␥, G␥ was isolated from the membrane fractions of 10 11 cells (Table 1).…”

“…GTT TTA AAA GAA AAT GAA ACC G C G Approximately 1.5 ϫ 10 5 phages from gt11 cDNA library, prepared from mRNA isolated at 2-4 h of development, were screened (Lilly et al, 1993). Fifty-two positive clones were detected and 4 independent clones were obtained after secondary and tertiary screens.…”

Selection of Gβ Subunits with Point Mutations That Fail to Activate Specific Signaling Pathways In Vivo: Dissecting Cellular Responses Mediated by a Heterotrimeric G Protein inDictyostelium discoideum

“…Stable expression of a G␤ subunit has been shown to require G␥ to form a G␤␥ dimer (Schmidt and Neer, 1991). Previous studies have demonstrated that the protein level of the D. discoideum G␤ does not change even when its mRNA is overexpressed Ͼ10 fold, presumably because excess G␤ proteins that do not form dimers with G␥ subunits are unstable (Lilly et al, 1993). We demonstrated that each G␤ subunit (SN1-SN5 alleles) was expressed at same level as a wild-type G␤, suggesting that each SN G␤ subunit can associate with a G␥ to form a stable G␤␥ dimer.…”

“…The new transformants showed the same characteristics as did the original mutants. To rule out the possibility that the phenotypes of the SN mutants were caused by an inadequate expression of the mutant G␤ subunits, we carried out immunoblot analysis using specific G␤ antiserum (Lilly et al, 1993). As shown in Figure 2, the SN1-SN5 cells expressed G␤ at the same level as wild-type cells, whereas SN6 cells expressed G␤ at a lower level.…”

“…In Dictyostelium discoideum, there is a single G␤ gene that is essential for the organism's developmental program. The G␤ null cells are viable but are unable to develop or differentiate because of an inability to regulate multiple signal transduction pathways (Lilly et al, 1993;Wu et al, 1995).…”