Identification and characterization of Csh3 as a SH3 protein that interacts with fission yeast Cap1

Schizosaccharomyces pombe Cap1 has been identified as the (adenylyl) cyclase associated protein. Cap1 was able to form homomers , and also binds actin. Cap1 localized to the growing tip, and this localization was dependent on the the P2 region in the middle domain. In a two-hybrid screening using cap1 as a bait, we isolated csh3, which encodes a protein of 296 amino acids with an SH3 domain and a proline/glutamine rich region. The binding of Csh3 and Cap1 was confirmed by in vivo pull down assays. Cooperative functions of Csh3 and Cap1 were observed. Deletion of both csh3 and cap1 resulted in heightened sensitivity to CaCl2, while disruption of either gene alone did not have any effect in this regard. In addition, over-expression of csh3 or cap1 alone did not affect cell growth, while over-expression of both genes resulted in growth retardation. Finally, while Csh3-GFP localized to the cytoplasm in wild-type cells, its localization was altered in cap1Δ cells, suggesting that the interaction between Csh3 and Cap1 controls the cellular localization of Csh3. These results demonstrate that Cap1 in S. pombe is a multifunctional protein that functions through dimerization (or multimerization) and interaction with other proteins including adenylyl cyclase, actin and a protein Csh3.