Expression of CEL2 and CEL4, Two Proteins from Agaricus Bisporus with Similarity to Fungal Cellobiohydrolase I and {beta}-mannanase, Respectively, is Regulated by the Carbon Source

1 Division of Life Sciences, King's College London, Campden Hill Road, London W8 7AH, UK 2 Amycel/Spawnmate, 777 Maher Court, PO Box 189, Watsonville, CA 95076-9014, USA 3 Plant Pathology and Microbiology Department, Horticulture Research International, Wellesbourne, Warwick CV35 9EF, UK ABSTRACT Two new cellulose-growth specific ( cel ) cDNAs, cel2 and cel4 , have been isolated from an Agaricus bisporus cDNA expression library by immunoscreening with an A. bisporus anti-‘endoglucanase’ antibody. The deduced amino acid sequences showed that both CEL2 and CEL4 proteins have a modular structure consisting of a fungal-type cellulose-binding domain (CBD) and a catalytic domain separated by a linker region rich in Pro, Ser and Thr. The CEL2 and CEL4 catalytic domains were homologous to fungal cellobiohydrolases (CBH) in family 7 and to fungal mannanases in family 5 of the glycosyl hydrolases, respectively. A previously isolated cDNA derived from a constitutive gene was also sequenced. The deduced amino acid sequence corresponded to 5-aminolaevulinic acid synthase (ALA), the first enzyme in the haem biosynthetic pathway, and was most similar to other fungal ALAs. RNA analysis showed that the expression of cel2 and cel4 genes was induced by cellulose and repressed by glucose, fructose and lactose. The soluble cellulose derivative CM-cellulose induced mRNA accumulation for cel1 but not cel2, cel3 or cel4. Mannitol, maltose, sorbitol and glycerol decreased cel2 and cel4 mRNA levels to different extents, cel1, cel2, cel3 and cel4 mRNAs all disappeared after the addition of glucose with apparent half-lives of less than 20 min. Whether cel mRNAs have short half-lives or glucose affects the stability of cel transcripts remains to be investigated. 4 Author for correspondence: Ernesto Yagüe. Tel: + 44 171 955 4083; Fax: +44 171 403 8883. Keywords: Agaricus bisporus, cellobiohydrolase, mannanase, gene regulation, 5-aminolaevulinic acid synthase Present address: Department of Experimental Pathology, UMDS, Guy's Hospital, London Bridge, London SE1 9RT, UK.