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Published on July 18, 2002; 10.1104/pp.002055

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Received December 22, 2001
Returned for revision February 3, 2002
Accepted April 7, 2002

A Thapsigargin-Sensitive Ca2+ Pump Is Present in the Pea Golgi Apparatus Membrane

Viviana R. Ordenes , Francisca C. Reyes , Daniel Wolff , and Ariel Orellana *

Instituto Milenio de Estudios Avanzados en Biología Celular y Biotecnología, Universidad de Chile, Casilla 653, Santiago, Chile (V.R.O., F.C.R., A.O.); Escuela de Graduados, Universidad de Concepción, Concepción, Chile (V.R.O.); and Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile (F.C.R., D.W., A.O.)

* Corresponding author; email: aorellan{at}uchile.cl.

The Golgi apparatus behaves as a bona fide calcium 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 Ca2+ accumulation mechanism in the plant cell Golgi apparatus. Toward this end, we measured Ca2+ 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 calcium uptake activity and the Golgi apparatus marker enzyme, xyloglucan-fucosyltransferase. The kinetic parameters obtained for this activity were: the rate of maximum Ca2+ uptake of 2.5 nmol mg min-1 and an apparent Km for Ca2+ of 209 nM. The ATP-dependent calcium uptake was strongly inhibited by vanadate (inhibitor concentration causing 50% inhibition [I50] = 126 µM) and cyclopiazonic acid (I50 = 0.36 nmol mg protein-1) and was not stimulated by calmodulin (1 µM). Addition of Cd2+ and Cu2+ at nanomolar concentration inhibited the calcium uptake, whereas Mn2+, Fe2+, and Co2+ had no significant effect. Interestingly, the active calcium uptake was inhibited by thapsigargin (apparent I50 = 88 nM), a well-known inhibitor of the endoplasmic reticulum and Golgi sarco-endoplasmic reticulum calcium ATPase from mammalian cells. A thapsigargin-sensitive calcium uptake activity was also detected in a cauliflower (Brassica oleracea) Golgi-enriched fraction, suggesting that other plants may also possess thapsigargin-sensitive Golgi calcium pumps. To our knowledge, this is the first report of a plant calcium pump activity that shows sensitivity to low concentrations of thapsigargin.




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