Identification of critical domains and putative partners for the Caenorhabditis elegans spindle component LIN-5.
Successful cell division requires proper assembly, placement and functioning of the spindle apparatus that segregates the chromosomes. The Caenorhabditis elegans gene lin-5 encodes a novel coiled-coil component of the spindle required for spindle positioning and chromosome segregation. To gain further insights into lin-5 function, we screened for dominant suppressors of the partial loss-of-function phenotype associated with the mutation lin-5(ev571ts ), and isolated 68 suppressing mutations. Eight out of the ten suppressors sequenced contained intragenic missense mutations immediately upstream of the lesion in lin-5(ev571ts ). These probably help to stabilize protein-protein interactions mediated by the coiled-coil domain. This domain was found to be required for binding to several putative LIN-5 interacting (LFI) proteins identified in yeast two-hybrid screens. Interestingly, interaction with the coiled-coil protein LFI-1 was specifically reduced by the lin-5(ev571ts ) mutation and restored by a representative intragenic suppressor mutation. Immunostaining experiments showed that LIN-5 and LFI-1 may co-localize around the kinetochore microtubules during metaphase, indicating potential interaction in vivo. The coiled-coil domain of LIN-5 was also found to mediate homodimerization, while the C-terminal region of LIN-5 was sufficient for interaction with GPR-1, a recently identified component of a LIN-5 spindle-regulatory complex. A single amino-acid substitution in the N-terminal region of LIN-5, encoded by the e1457 allele, abolished all LIN-5 interactions. Taken together, our results indicate that the spindle functions of LIN-5 depend on interactions with multiple protein partners, and that these interactions are mediated through several different domains of LIN-5.
Fisk Green
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Massachusetts General Hospital Cancer Center (Bldg. 149), Harvard Medical School, Charlestown, MA 02129, USA.
Lorson
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Walhout
A J M
AJ
Vidal
M
M
van den Heuvel
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S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000737:chemistry*; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry* / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000737:chemistry*; D017238:Genes, Helminth; D008938:Mitosis; D008969:Molecular Sequence Data; D009154:Mutation; D008941:Spindle Apparatus / Q000737:chemistry*; D020798:Two-Hybrid System Techniques
D029742:Caenorhabditis elegans Proteins; D018797:Cell Cycle Proteins; C475458:G protein regulator 1, C elegans; C475459:G protein regulator 2, C elegans; C404808:lin-5 protein, C elegans