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 activity in resting mycelia of Trichoderma reesei. The inhibition by the ionophore was reversed by the addition of Ca2+ ions. A similar inhibitory effect was obtained by the addition of the calmodulin inhibitors, trifluoroperazine, chlorpromazine and quinacrine, hence suggesting that the observed effect of Ca2+ on xylanase formation occurred via calmodulin. The inhibition of xylanase formation by trifluoroperazine was accompanied by an inhibition of formation of the xyn2 transcript, and of the hph (hygromycin B-phosphotransferase-encoding) gene when fused downstream of the 5'-regulatory signals of the T. reesei xyn2 gene, indicating that calmodulin is required for xyn2 induction. At trifluoroperazine concentrations, which inhibited extracellular xylanase formation 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 light vesicular fractions. This accumulated XYN II protein had the same Mr as the secreted, extracellular enzyme, indicating that it had already passed Golgi-located preprotein processing. Trifluoroperazine also specifically interfered with the endogenous, Ca2+-dependent phosphorylation of a 20-kDa protein, which was predominantly observed in cell-free extracts from mycelia growing on xylan. From these data, we conclude 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 the secretory pathway. We also suggest that at least one of these two steps may be mediated via Ca2+-calmodulin-dependent phosphorylation.
Abteilung für Mikrobielle Biochemie, Institut für Biochemische Technologie und Mikrobiologie, TU Wien, Getreidemarkt 9/172-5, A-1060 Vienna, Austria.
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D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000037:antagonists & inhibitors*; D002147:Calmodulin / Q000037:antagonists & inhibitors*; D002614:Chelating Agents / Q000494:pharmacology; D002746:Chlorpromazine / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D007811:Lanthanum / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism; D011796:Quinacrine / Q000494:pharmacology; D013347:Subcellular Fractions / Q000201:enzymology; D014158:Transcription, Genetic; D014242:Trichoderma / Q000187:drug effects / Q000201:enzymology*; D014268:Trifluoperazine / Q000494:pharmacology; D043325:Xylan Endo-1,3-beta-Xylosidase; D014995:Xylosidases / Q000096:biosynthesis* / Q000235:genetics
D002147:Calmodulin; D002614:Chelating Agents; D007476:Ionophores; C028521:lanthanum chloride; D014268:Trifluoperazine; D000001:Calcimycin; D004533:Egtazic Acid; D007811:Lanthanum; D011494:Protein Kinases; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase; D011796:Quinacrine; D002118:Calcium; D002746:Chlorpromazine