Inhibition of the proteasome strongly affects cadmium stimulated laccase activity in Trametes versicolor

Most proteins in eukaryotic cells are degraded by a highly selective non-lysosomal pathway that requires ATP and a large multicatalytic proteinase complex known as the 26S proteasome. In the present study, we evaluated the possibility that the proteasome-mediated pathway is involved in the regulation of laccase production by the efficient lignin-degrading basidiomycete Trametes versicolor in response to cadmium. These studies were performed using MG132 and lactacystin beta-lactone as specific proteasome inhibitors separately added to the culture medium of 7-day-old mycelia of T. versicolor at the start of incubation with 10-200 muM CdCl(2). We found that Cd(2+) stimulated laccase activity at all concentrations tested. The highest increase was observed at 100 muM Cd(2+), where laccase activity was three to fivefold higher than in Cd(2+)-free cultures. Blocking of proteasome function in Cd(2+)-supplemented cultures resulted in the considerably lower laccase activity in comparison to controls with no proteasomal inhibitor added. The decline of extracellular laccase activity triggered by the proteasome inhibitors was especially significant in the case of cultures with 100 muM Cd(2+), where around seven and threefold lost of laccase activity was observed for MG132 and lactacystin beta-lactone, respectively. Similar findings were obtained for intracellular laccase. In contrast to Cd(2+)-supplemented cultures, no significant change in laccase activity could be detected for Cd(2+)-free cultures after exposure to the proteasome inhibitors. Effects observed with chloroquine, the inhibitor of lysosomal proteolysis, added to T. versicolor cultures were markedly different from those found in the case of the proteasome inhibitors. We also showed that addition of Cd(2+) to growing cultures of T. versicolor did not significantly affect proteasome activities detected in high molecular (above 500 kDa) fractions of mycelial extracts. Our results strongly support the interpretation that the proteasome-mediated proteolytic pathway plays an important role in the regulation of T. versicolor laccase activity in response to Cd(2+).