HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 283, Issue 31, 21550-21558, August 1, 2008

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 283/31/21550    most recent M803023200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Johnston, C. A. Articles by Willard, F. S. Search for Related Content PubMed PubMed Citation Articles by Johnston, C. A. Articles by Willard, F. S. Social Bookmarking                      What's this?

Structural Determinants Underlying the Temperature-sensitive Nature of a G Mutant in Asymmetric Cell Division of Caenorhabditis elegans^*

Christopher A. Johnston¹², Katayoun Afshar¹, Jason T. Snyder, Gregory G. Tall^¶3, Pierre Gönczy^||, David P. Siderovski^**4, and Francis S. Willard

From the Department of Pharmacology and ^**Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599, the Swiss Institute for Experimental Cancer Research and ^||School of Life Sciences, Swiss Federal Institute of Technology, 1066 Lausanne, Switzerland, and the ^¶Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642

Heterotrimeric G-proteins are integral to a conserved regulatory module that influences metazoan asymmetric cell division (ACD). In the Caenorhabditis elegans zygote, GOA-1 (G_o) and GPA-16 (G_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 G_i1(G202D) as a model in biochemical analyses, we demonstrate that high temperature induces instability of the mutant G. At the permissive temperature, the mutant G 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 G 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 G subunit function in ACD.

Received for publication, April 21, 2008 , and in revised form, June 2, 2008.

The atomic coordinates and structure factors (code 2EBC) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported, in whole or in part, by National Institutes of Health Grant GM074268 (to D. P. S.). This work was also supported by Swiss National Science Foundation Grant 3100A0-102087 (to P. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables 1 and 2 and Movies S1–S6.

¹ Both authors contributed equally to this work.

² Supported by National Institutes of Health Fellowship GM076944.

³ Supported by American Heart Association Grant 0325033Y.

⁴ To whom correspondence should be addressed: 1106 M. E. Jones Bldg., CB 7365, Chapel Hill, NC 27599. Tel.: 919-843-9363; Fax: 919-966-5640; E-mail: dsiderov{at}med.unc.edu.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				F. S. Willard, Z. Zheng, J. Guo, G. J. Digby, A. J. Kimple, J. M. Conley, C. A. Johnston, D. Bosch, M. D. Willard, V. J. Watts, et al. A Point Mutation to G{alpha}i Selectively Blocks GoLoco Motif Binding: DIRECT EVIDENCE FOR G{alpha}{middle dot}GoLoco COMPLEXES IN MITOTIC SPINDLE DYNAMICS J. Biol. Chem., December 26, 2008; 283(52): 36698 - 36710. [Abstract] [Full Text] [PDF]