RESUMO
Chitin deacetylases are required for spore wall rigidity in Saccharomyces cerevisiae. Two chitin deacetylase genes (CDA1 and CDA2) have been identified in yeast. In this report we studied the biochemical properties of the chitin deacetylases encoded by CDA1 and CDA2 and we show how their elimination directly affects the ascospore wall assembly.
Assuntos
Amidoidrolases/fisiologia , Parede Celular/enzimologia , Saccharomyces cerevisiae/enzimologia , Ácido Acético/farmacologia , Amidoidrolases/genética , Divisão Celular/genética , Parede Celular/fisiologia , Regulação Fúngica da Expressão Gênica , Glucosamina/metabolismo , Isoenzimas , Mutagênese , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Fatores de Tempo , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
The formation of the ascospore wall of Saccharomyces cerevisiae requires the coordinate activity of enzymes involved in the biosynthesis of its components such as chitosan, the deacetylated form of chitin. We have cloned the CDA1 and CDA2 genes which together account for the total chitin deacetylase activity of the organism. We have shown that expression of these genes is restricted to a distinct time period during sporulation. The two genes are functionally redundant, each contributing equally to the total chitin deacetylase activity. Diploids disrupted for both genes sporulate as efficiently as wild type cells, and the resulting mutant spores are viable under standard laboratory conditions. However, they fail to emit the natural fluorescence of yeast spores imparted by the dityrosine residues of the outermost ascospore wall layer. Moreover, mutant spores are relatively sensitive to hydrolytic enzymes, ether, and heat shock, a fact that underscores the importance of the CDA genes for the proper formation of the ascospore wall.
Assuntos
Amidoidrolases/genética , Isoenzimas/genética , Saccharomyces cerevisiae/fisiologia , Sequência de Aminoácidos , Northern Blotting , Parede Celular/genética , Regulação Enzimológica da Expressão Gênica , Dados de Sequência Molecular , Saccharomyces cerevisiae/enzimologia , Alinhamento de Sequência , Esporos Fúngicos/enzimologia , Tirosina/análogos & derivadosRESUMO
Two alcohol dehydrogenase isozymes, namely ADH-1 and ADH-2 from Ceratitis capitata were purified to homogeneity and further characterized. After ammonium sulphate precipitation from an extract of whole third instar larvae, the two isozymes were separated by ion exchange chromatography on Q-Sepharose. A combination of affinity chromatography, gel filtration and ion exchange chromatography was then used to purify each isozyme (50 and 57 times with 53 and 58% yields, for ADH-1 and -2 respectively). A crucial step for obtaining homogeneous enzyme preparations was affinity chromatography on Cibacron Blue Sepharose coupled with specific elution with NAD. Each of the isozymes is a dimer with subunit molecular weight of approximately 27 kDa. Both isozymes show a pH optimum of 9.6. ADH-1 proved to be immunochemically similar to ADH-2 when tested by Western blot analysis using polyclonal antibodies raised against ADH-1. While crude extracts of Dacus oleae ADH cross-react with these antibodies, no cross reactivity was observed with Drosophila melanogaster extracts. The sequence of a 22-residue peptide from ADH-1 was determined and showed 36% identity with residues 26-47 of the Drosophila melanogaster ADH sequence. Both the sizes of the purified proteins and the observed sequence similarity between ADH-1 and Drosophila ADH strongly suggest that the medfly ADH isozymes belong to the family of short chain dehydrogenases.