D. Pitt et Jc. Barnes, CALCIUM HOMEOSTASIS, SIGNALING AND PROTEIN-PHOSPHORYLATION DURING CALCIUM-INDUCED CONIDIATION IN PENICILLIUM-NOTATUM, Journal of General Microbiology, 139, 1993, pp. 3053-3063
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.