The essential mitotic target of calmodulin is the 110-kilodalton component of the spindle pole body in Saccharomyces cerevisiae.
Two independent methods identified the spindle pole body component Nuf1p/Spc110p as the essential mitotic target of calmodulin. Extragenic suppressors of cmd1-1 were isolated and found to define three loci, XCM1, XCM2, and XCM3 (extragenic suppressor of cmd1-1). The gene encoding a dominant suppressor allele of XCM1 was cloned. On the basis of DNA sequence analysis, genetic cosegregation, and mutational analysis, XCM1 was identified as NUF1/SPC110. Independently, a C-terminal portion of Nuf1p/Spc110p, amino acid residues 828 to 944, was isolated as a calmodulin-binding protein by the two-hybrid system. As assayed by the two-hybrid system, Nuf1p/Spc110p interacts with wild-type calmodulin and triple-mutant calmodulins defective in binding Ca2+ but not with two mutant calmodulins that confer a temperature-sensitive phenotype. Deletion analysis by the two-hybrid system mapped the calmodulin-binding site of Nuf1p/Spc110p to amino acid residues 900 to 927. Direct binding between calmodulin and Nuf1p/Spc110p was demonstrated by a modified gel overlay assay. Furthermore, indirect immunofluorescence with fixation procedures known to aid visualization of spindle pole body components localized calmodulin to the spindle pole body. Sequence analysis of five suppressor alleles of NUF1/SPC110 indicated that suppression of cmd1-1 occurs by C-terminal truncation of Nuf1p/Spc110p at amino acid residues 856, 863, or 881, thereby removing the calmodulin-binding site.
Geiser
J R
JR
Department of Biochemistry, University of Washington, Seattle 98195.
Sundberg
H A
HA
Chang
B H
BH
Muller
E G
EG
Davis
T N
TN
D001665:Binding Sites; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D005800:Genes, Fungal; D016153:Genes, Suppressor; D007150:Immunohistochemistry; D008938:Mitosis / Q000235:genetics / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D008941:Spindle Apparatus / Q000378:metabolism*
D002147:Calmodulin; D004271:DNA, Fungal; D005656:Fungal Proteins