Structural determinants underlying the temperature-sensitive nature of a Gα mutant in asymmetric cell division of Caenorhabditis elegans

Christopher A. Johnston, Katayoun Afshar, Jason T. Snyder, Gregory G. Tall, Pierre Gönczy, David P. Siderovski, Francis S. Willard

Research output: Contribution to journalArticle

12 Scopus citations

Abstract

Heterotrimeric G-proteins are integral to a conserved regulatory module that influences metazoan asymmetric cell division (ACD). In the Caenorhabditis elegans zygote, GOA-1 (Gαo) and GPA-16 (Gαi) 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 Gαi1(G202D) as a model in biochemical analyses, we demonstrate that high temperature induces instability of the mutant Gα. At the permissive temperature, the mutant Gα 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 Gα 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 wild type. 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 wild type 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 Gα subunit function in ACD.

Original languageEnglish
Pages (from-to)21550-21558
Number of pages9
JournalJournal of Biological Chemistry
Volume283
Issue number31
DOIs
StatePublished - 1 Aug 2008

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