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

Schizosaccharomyces pombe Cap1 has been identified as the (adenylyl) cyclase-associated protein. Cap1 was able to bind Cap1 itself and actin. Cap1 localized at the growing tip, and this localization was dependent on the Cap1 P2 region. In a two-hybrid screening using cap1 as 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 Schizo. pombe is a multifunctional protein that functions through interaction with Cap1 itself and other proteins including adenylyl cyclase, actin and Csh3.

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