Heterotrimeric G-proteins are integral to a conserved regulatory module that influences metazoan asymmetric cell division (ACD). In the Caenorhabditis elegans zygote, GOA-1 (Galphao) and GPA-16 (Galphai) are involved in generating forces that pull on astral microtubules and position the spindle asymmetrically. GPA-16 function has been analyzed in vivo owing notably to a temperature-sensitive allele
gpa-16(
it143) which, at the restrictive temperature, results in spindle orientation defects in early embryos. Here, we identify the structural basis of
gpa-16(
it143) which encodes a point mutation (G202D) in the switch-II region of GPA-16. Using Galphai1(G202D) as a model in biochemical analyses, we demonstrate that high temperature induces instability of the mutant Galpha. At the permissive temperature, the mutant Galpha was stable upon GTP binding, but switch II rearrangement was compromised, as were activation state-selective interactions with regulators involved in ACD, including GoLoco motifs, RGS proteins, and RIC-8. We solved the crystal structure of the mutant Galpha bound to GDP, which indicates a unique switch II conformation as well as steric constraints that suggest activated GPA 16[
it143] is destabilized relative to wildtype. Spindle severing in
gpa-16(
it143) embryos revealed that pulling forces are symmetric and markedly diminished at the restrictive temperature. Interestingly, pulling forces are asymmetric and generally similar in magnitude to wildtype at the permissive temperature despite defects in the structure of GPA-16[
it143]. These normal pulling forces in
gpa-16(
it143) embryos at the permissive temperature were attributable to GOA-1 function, underscoring a complex interplay of Galpha subunit function in ACD.