CALCIUM HOMEOSTASIS, SIGNALING AND PROTEIN-PHOSPHORYLATION DURING CALCIUM-INDUCED CONIDIATION IN PENICILLIUM-NOTATUM

Cytosolic free calcium concentration [Ca2+], of protoplasts from Penic illium notatum was measured using the permeant acetoxy ester (quin-2-A M) of the calcium-chelating fluorescent dye quin-2. Low uptake of the ester occurred at pH 5.8-7.0 and its subsequent hydrolysis was low. Up take was promoted by an external pH of 5.0 and significant hydrolysis to quin-2 achieved by adjustment of the internal pH to 7.2, which was near the optimum of the carboxylic esterases responsible for the hydro lysis. Uptake of Ca2+ was biphasic with the average cell calcium conce ntration of protoplasts increasing from an initial value of 2 mu mol t o 50 mu mol (kg cell water)(-1), during attainment of the steady state after 30 min, at which time [Ca2+], was unchanged at 20 nM but increa sed to 182 nm at 2-6 h exposure to 2.5 mM-Ca2+. Broadly similar change s in [Ca2+], were found in protoplasts derived from mycelium samples e xposed to Ca2+ over the same period of time. The location of Ca2+ was determined in subfractionated organelles and characterized using enzym e markers and electron microscopy. In 32 h mycelium preloaded with Ca2 + for 6 h, Ca2+ was located principally in the mitochondria with lower concentrations associated with the endoplasmic reticulum, Golgi, vacu oles and plasma membrane components. Calcium was not released by inosi tol 1,4,5-trisphosphate or the calcium ionophore A23187 from any subce llular fractions obtained from mycelium on Percoll gradients, nor from preparations of vacuoles or plasmalemma vesicles, except in the case of mitochondria where rapid release of the ion was achieved by the add ition of 2-5 mu M-A23187. The anti-calmodulin agent calmidazolium (R24 571) greatly reduced sporulation when addition preceded that of Ca2+. Calcium-induced cultures showed massive novel protein phosphorylation 2 h after addition of the ion which was virtually eliminated by R24571 , whilst 1 h and 4-6 h protein phosphorylations, which were also prese nt to some degree in vegetative controls, were substantially reduced. Two-dimensional SDS-PAGE analysis of phosphoproteins confirmed that Ca 2+-induced mycelium had enhanced capacity for calmodulin-mediated phos phorylation relative to corresponding vegetative cells and that comple x differential changes in such phosphorylations occurred during Ca2+-i nduction of the sporulation process.