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Identification of a CAP (adenylyl-cyclase-associated protein) homologous gene in Lentinus edodes and its functional complementation of yeast CAP mutants

Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, Matsue 690, Japan
Department of Life Science, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226, Japan
Research Institute of Molecular Genetics, Shimane University, Matsue 690, Japan

ABSTRACT

The adenylyl-cyclase-associated protein, CAP, was originally identified in yeasts as a protein that functions in both signal transduction and cytoskeletal organization. This paper reports the identification of a cDNA and genomic DNA that encodes a CAP homologue from the mushroom Lentinus edodes. The L. edodes cap gene contains eight introns and an ORF encoding a 518 amino acid protein. The L. edodes CAP is 35.5% and 40.9% identical at the amino acid level with Saccharomyces cerevisiae CAP and Schizosaccharomyces pombe CAP, respectively. The C-terminal domain shows greater homology (39-46% identity) with yeast CAPs than does the N-terminal domain (27-35% identity). Southern blotting and Northern blotting results suggest that L. edodes cap is a single-copy gene and uniformly expressed. Expression of the L. edodes CAP in both Schiz. pombe and Sacch. cerevisiae complemented defects associated with the loss of the C-terminal domain function of the endogenous CAP. By using a yeast two-hybrid assay, an interaction was demonstrated between the L. edodes CAP and Schiz. pombe actin. This result and the functional complementation test indicate that CAP from L. edodes has a conserved C-terminal domain function.

^*Author for correspondence: Makoto Kawamukai. Tel: +81 852 32 6587. Fax: +81 852 32 6499. e-mail: kawamuka@life.shimane-u.ac.jp

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