INSITU LOCALIZATION OF THE SECRETION OF LIGNIN PEROXIDASES IN COLONIES OF PHANEROCHAETE-CHRYSOSPORIUM USING A SANDWICHED MODE OF CULTURE

Protein secretion and growth were investigated in Phanerochaete chrysosporium by using cultures sandwiched between perforated polycarbonate membranes. Labelling of colonies with radioactive N-acetylglucosamine and L-methionine indicated a close correlation between growth and general protein secretion, even in a central area of the colony secreting the idiophase enzymes lignin peroxidase (LiP) and manganese-dependent lignin peroxidase (MnP). Comparison of the sites of release into the medium of newly synthesized proteins and immuno-detected lignin peroxidases suggested that diffusion of the enzymes from the walls was a limiting step in the release of peroxidases into the medium. Microautoradiography of colonies exposed to N-acetyl[H-3]glucosamine revealed the apical growth of thin hyphae and branches (4 to 5 mum diameter on average) in the central secreting area. These secondary hyphae showed peroxidase activity and reacted with lignin peroxidase antibodies. Although it was not possible to directly visualize secretion at hyphal tips, the results suggest that peroxidases (LiP and MnP) are initially secreted at the apex of secondary growing hyphae and later slowly released into the surrounding medium.