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  4. Structural determinants underlying the temperature-sensitive nature of a Galpha mutant in asymmetric cell division of Caenorhabditis elegans
 
research article

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

Johnston, Christopher A.
•
Afshar, Katayoun  
•
Snyder, Jason T.
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2008
Journal of Biological Chemistry

Heterotrimeric G-proteins are integral to a conserved regulatory module that influences metazoan asymmetric cell division (ACD). In the Caenorhabditis elegans zygote, GOA-1 (Galpha(o)) and GPA-16 (Galpha(i)) 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 Galpha(i1)(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 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 Galpha subunit function in ACD

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Type
research article
DOI
10.1074/jbc.M803023200
Web of Science ID

WOS:000257898800028

Author(s)
Johnston, Christopher A.
Afshar, Katayoun  
Snyder, Jason T.
Tall, Gregory G.
Gönczy, Pierre  
Siderovski, David P.
Willard, Francis S.
Date Issued

2008

Published in
Journal of Biological Chemistry
Volume

283

Issue

31

Start page

21550

End page

21558

Note

Author address: Department of Pharmacology and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA.

Editorial or Peer reviewed

REVIEWED

Written at

EPFL

EPFL units
UPGON  
Available on Infoscience
August 31, 2008
Use this identifier to reference this record
https://infoscience.epfl.ch/handle/20.500.14299/27668
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