An association of trehalase with the innermost wall (endosporium) of ascospores of Neurospora is suggested, because this enzyme could be lyophilized in the presence of various wall components and heated in this dried state at 65 C without loss of activity. Ground ascospore walls, purified mycelial walls, a wall fraction consisting of protein, glucan and polygalactosamine, or bovine serum albumin stabilize trehalase under these conditions. No other substances tested protected as well as the above materials. Immunofluorescent labeling of trehalase shows that it is localized in the endosporium. Therefore, it is most probable that in dormant ascospores of Neurospora, trehalase, and its substrate, trehalose, are physically separated. Trehalose is located in the cytoplasm, whereas trehalase resides within the protein and carbohydrate matrix of the innermost major cell wall layer of the ascospore. The association with the cell wall protects the enzyme against the heating which is necessary to activate germination. Activation, whether by heat or chemical treatment (furfural), probably involves an increase in the permeability of the ascospore plasma membrane allowing trehalose to diffuse to the vicinity of its hydrolase, thereby providing the energy and intermediates for germination.

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