A Novel Mechanism of Intragenic Complementation between Phe to Ala Calmodulin Mutations

Abstract

Calmodulin (CaM) performs essential functions in cell proliferation in Saccharomyces cerevisiae. Previously, we isolated fourteen temperature-sensitive Phe-to-Ala mutations of the CaM-encoding gene CMD1. These mutations were classified into four intragenic complementation groups, suggesting that each group represents a loss of CaM interaction with its specific essential target protein. Nuf1p/Spc110p, one of the essential targets, is a spindle pole body component that is required for proper mitosis. We investigated which intragenic complementation group of CaM represents the malfunction of Nuf1p. Immunoprecipitation analysis showed that two cmd1 mutations belonging to two distinct intragenic complementation groups had the most severely impaired complex formation with Nuf1p at the restrictive temperature. The temperature-sensitive growth of these cmd1 mutants was suppressed by a CaM-independent dominant allele of NUF1. Additionally, these mutants displayed characteristic mitotic defects: an increased ratio of artificial chromosome loss, which could be suppressed by the CaM-independent dominant allele of NUF1, and aberrant microtubule structures. These results indicate that these cmd1 mutants display the temperature-sensitive growth due to the compromised interaction with Nuf1p. However, the interaction was restored in a heterozygous diploid of the two cmd1 alleles, suggesting that intragenic complementation between these cmd1 alleles occurs by a novel mechanism, whereby co-presence of both mutant proteins rescues the interaction with Nuf1p.