CA2-CALMODULIN ANTAGONISTS INTERFERE WITH XYLANASE FORMATION AND SECRETION IN TRICHODERMA-REESEI()

The addition of Ca2+-antagonizers (La2+), Ca2+-ionophores (A23187) and Ca2+-complexing agents (EGTA) inhibited the formation of xylanase act ivity in resting mycelia of Trichoderma reesei. The inhibition by the ionophore was reversed by the addition of Ca2+ ions. A similar inhibit ory effect was obtained by the addition of the calmodulin inhibitors, trifluoroperazine, chlorpromazine and quinacrine, hence suggesting tha t the observed effect of Ca2+ on xylanase formation occurred via calmo dulin. The inhibition of xylanase formation by trifluoroperazine was a ccompanied by an inhibition of formation of the xyn2 transcript, and o f the hph (hygromycin B-phosphotransferase-encoding) gene when fused d ownstream of the 5'-regulatory signals of the T. reesei xyn2 gene, ind icating that calmodulin is required for xyn2 induction. At trifluorope razine concentrations, which inhibited extracellular xylanase formatio n only slightly (about 30%), the cell-free extracts exhibited slightly increased xylanase activities. Subcellular fractionation showed that in these mycelia, the XYN II protein was distributed over a range of l ight vesicular fractions. This accumulated XYN II protein had the same M-r as the secreted, extracellular enzyme, indicating that it had alr eady passed Golgi-located preprotein processing. Trifluoroperazine als o specifically interfered with the endogenous, Ca2+-dependent phosphor ylation of a 20-kDa protein, which was predominantly observed in cell- free extracts from mycelia growing on xylan. From these data, we concl ude that calmodulin is required for xylanase II formation by T. reesei both at a transcriptional level as well as at a post-Golgi step of th e secretory pathway. We also suggest that at least one of these two st eps may be mediated via Ca2+-calmodulin-dependent phosphorylation. (C) 1998 Elsevier Science B.V. All rights reserved.