Role of calmodulin and Spc110p interaction in the proper assembly of spindle pole body compenents.
Previously we demonstrated that calmodulin binds to the carboxy terminus of Spc110p, an essential component of the Saccharomyces cerevisiae spindle pole body (SPB), and that this interaction is required for chromosome segregation. Immunoelectron microscopy presented here shows that calmodulin and thus the carboxy terminus of Spc110p localize to the central plaque. We created temperature-sensitive SPC110 mutations by combining PCR mutagenesis with a plasmid shuffle strategy. The temperature-sensitive allele spc110-220 differs from wild type at two sites. The cysteine 911 to arginine mutation resides in the calmodulin-binding site and alone confers a temperature-sensitive phenotype. Calmodulin overproduction suppresses the temperature sensitivity of spc110-220. Furthermore, calmodulin levels at the SPB decrease in the mutant cells at the restrictive temperature. Thus, calmodulin binding to Spc110-220p is defective at the nonpermissive temperature. Synchronized mutant cells incubated at the nonpermissive temperature arrest as large budded cells with a G2 content of DNA and suffer considerable lethality. Immunofluorescent staining demonstrates failure of nuclear DNA segregation and breakage of many spindles. Electron microscopy reveals an aberrant nuclear structure, the intranuclear microtubule organizer (IMO), that differs from a SPB but serves as a center of microtubule organization. The IMO appears during nascent SPB formation and disappears after SPB separation. The IMO contains both the 90-kD and the mutant 110-kD SPB components. Our results suggest that disruption of the calmodulin Spc110p interaction leads to the aberrant assembly of SPB components into the IMO, which in turn perturbs spindle formation.
Sundberg
H A
HA
Department of Biochemistry, University of Washington, Seattle, 98195-7350. USA.
Goetsch
L
L
Byers
B
B
Davis
T N
TN
D001665:Binding Sites; D002147:Calmodulin / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins; D002453:Cell Cycle / Q000502:physiology; D002467:Cell Nucleus / Q000737:chemistry; D015825:Chromosomes, Fungal; D003598:Cytoskeletal Proteins; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D008870:Microtubules / Q000737:chemistry* / Q000648:ultrastructure; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins; D008941:Spindle Apparatus / Q000737:chemistry* / Q000648:ultrastructure; D013489:Suppression, Genetic; D013696:Temperature
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D003598:Cytoskeletal Proteins; D005656:Fungal Proteins; D009687:Nuclear Proteins; C073277:SPC110 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins