Identification of critical domains and putative partners for the Caenorhabditis elegans spindle component LIN-5
Program in Gene Function and Expression; Program in Molecular Medicine
Medical Subject Headings
Amino Acid Sequence; Animals; Base Sequence; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Cell Cycle Proteins; *Genes, Helminth; Mitosis; Mitotic Spindle Apparatus; Molecular Sequence Data; Mutation; Two-Hybrid System Techniques
Genetics and Genomics
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.
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Citation: Mol Genet Genomics. 2004 Jun;271(5):532-44. Epub 2004 May 12. Link to article on publisher's site
Green, Ridgely Fisk; Lorson, Monique A.; Walhout, Albertha J. M.; Vidal, Marc; and van den Heuvel, Sander, "Identification of critical domains and putative partners for the Caenorhabditis elegans spindle component LIN-5" (2004). Program in Gene Function and Expression Publications and Presentations. Paper 18.