Interaction with calmodulin is required for the function of Spc110p, an essential component of the yeast spindle pole body.
NUF1/SPC110, encoding a nuclear filament-related protein which is a component of the yeast spindle pole body (SPB), has been identified in a screen designed to isolate genes encoding targets of yeast calmodulin. Spc110p interacts with calmodulin by two different criteria and the calmodulin interacting region has been localized within the C-terminus of the protein. Point mutations between residues 898 and 917 further define the calmodulin binding site within this region. Mutations in this domain which abolish calmodulin binding in vitro prevent Spc110p function in vivo, demonstrating that calmodulin binding by Spc110p has important functional consequences. In keeping with a role for calmodulin in Spc110p function, we show that calmodulin localizes to the yeast SPB when cells are prepared under appropriate conditions. Non-functional mutant Spc110 proteins which cannot bind calmodulin are present at lowered steady-state levels in the cell; when their level is increased by elevated gene dosage, partial recovery of Spc110p function is seen. Overexpression of calmodulin suppresses the defect(s) associated with the mutant Spc110 proteins, supporting the notion that Spc110p stability is a consequence of its ability to bind calmodulin and pointing to a direct role for calmodulin in Spc110p function.
Stirling
D A
DA
Department of Biochemistry, The University, Dundee, UK.
Welch
K A
KA
Stark
M J
MJ
D000483:Alleles; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins; D002451:Cell Compartmentation; D002453:Cell Cycle / Q000502:physiology; D003598:Cytoskeletal Proteins; D004252:DNA Mutational Analysis; D005455:Fluorescent Antibody Technique; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal / Q000235:genetics*; D015698:Genomic Library; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins; D008941:Spindle Apparatus / Q000502:physiology*
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D003598:Cytoskeletal Proteins; D005656:Fungal Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; C073277:SPC110 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D002118:Calcium