A thapsigargin-sensitive Ca(2+) pump is present in the pea Golgi apparatus membrane.
The Golgi apparatus behaves as a bona fide Ca(2+) store in animal cells and yeast (Saccharomyces cerevisiae); however, it is not known whether this organelle plays a similar role in plant cells. In this work, we investigated the presence of an active Ca(2+) accumulation mechanism in the plant cell Golgi apparatus. Toward this end, we measured Ca(2+) uptake in subcellular fractions isolated from the elongating zone of etiolated pea (Pisum sativum) epicotyls. Separation of organelles using sucrose gradients showed a strong correlation between the distribution of an ATP-dependent Ca(2+) uptake activity and the Golgi apparatus marker enzyme, xyloglucan-fucosyltransferase. The kinetic parameters obtained for this activity were: the rate of maximum Ca(2+) uptake of 2.5 nmol mg min(-1) and an apparent K(m) for Ca(2+) of 209 nM. The ATP-dependent Ca(2+) uptake was strongly inhibited by vanadate (inhibitor concentration causing 50% inhibition [I(50)] = 126 microM) and cyclopiazonic acid (I(50) = 0.36 nmol mg protein(-1)) and was not stimulated by calmodulin (1 microM). Addition of Cd(2+) and Cu(2+) at nanomolar concentration inhibited the Ca(2+) uptake, whereas Mn(2+), Fe(2+), and Co(2+) had no significant effect. Interestingly, the active calcium uptake was inhibited by thapsigargin (apparent I(50) = 88 nM), a well-known inhibitor of the endoplasmic reticulum and Golgi sarco-endoplasmic reticulum Ca(2+) ATPase from mammalian cells. A thapsigargin-sensitive Ca(2+) uptake activity was also detected in a cauliflower (Brassica oleracea) Golgi-enriched fraction, suggesting that other plants may also possess thapsigargin-sensitive Golgi Ca(2+) pumps. To our knowledge, this is the first report of a plant Ca(2+) pump activity that shows sensitivity to low concentrations of thapsigargin.
Instituto Milenio de Estudios Avanzados en Biología Celular y Biotecnología, Universidad de Chile, Casilla 653, Santiago, Chile.
Viviana R
VR
Ordenes
Francisca C
FC
Reyes
Daniel
D
Wolff
Ariel
A
Orellana
D000255:Adenosine Triphosphate / Q000378:metabolism; D001937:Brassica / Q000187:drug effects / Q000502:physiology; D002104:Cadmium / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000187:drug effects / Q000378:metabolism*; D002147:Calmodulin / Q000494:pharmacology; D003035:Cobalt / Q000378:metabolism; D003300:Copper / Q000494:pharmacology; D005647:Fucosyltransferases / Q000378:metabolism; D006056:Golgi Apparatus / Q000187:drug effects / Q000201:enzymology*; D007211:Indoles / Q000494:pharmacology; D007425:Intracellular Membranes / Q000187:drug effects / Q000201:enzymology*; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D018532:Pisum sativum / Q000187:drug effects / Q000201:enzymology*; D019284:Thapsigargin / Q000494:pharmacology*; D014638:Vanadates / Q000494:pharmacology
D002147:Calmodulin; D007211:Indoles; D002104:Cadmium; D003035:Cobalt; D014638:Vanadates; D008345:Manganese; D019284:Thapsigargin; D003300:Copper; D000255:Adenosine Triphosphate; D007501:Iron; D005647:Fucosyltransferases; C070983:xyloglucan 2-fucosyltransferase; D000252:Calcium-Transporting ATPases; D002118:Calcium; C000543:cyclopiazonic acid