V‐ AND P‐TYPE Ca²⁺‐STIMULATED ATPases IN A CALCIFYING STRAIN OF PLEUROCHRYSIS SP. (HAPTOPHYCEAE)

Abstract

Coccolithophorids are marine unicellular algae characterized by their ability to carry out controlled, subcellular calcification. The biochemical and kinetic features of membrane-bound Ca 2ϩ -stimulated ATPases have been examined. Membranes and organelles from axenic cultures of Pleurochrysis sp. (CCMP299) were isolated by means of sucrose density centrifugation. High levels of Ca 2ϩ -stimulated ATPase were detected in chloroplasts, Golgi apparatus, plasma membrane, and coccolith vesicles. The sensitivity of the enzyme activity in the organelles and membranes was assessed with pharmacologic agents that are known to be specific for the several isoforms of Ca 2ϩ -stimulated ATPase. The Ca 2ϩ -stimulated ATPase activity in the Golgi and coccolith vesicle preparations was sensitive to nitrate, thiocyanate, and sodium azide and insensitive to vanadate, cyclopiazonic acid, and thapsigargin. ATPdependent H ϩ movement, but not 45 Ca 2ϩ transport, across the coccolith vesicle was demonstrated. The Ca 2ϩ -stimulated ATPase in the plasma membrane preparation was sensitive to vanadate. ATP-dependent, vanadate-sensitive efflux of 45 Ca 2ϩ was demonstrated for microsomal material derived from gradient-isolated plasma membrane. Polypeptides from isolated Golgi and coccolith vesicle preparations cross-reacted to an antibody raised against a subunit of the oat root proton pump, whereas polypeptides from the chloroplast preparations did not cross-react. These findings show that a V-type Ca 2ϩ -stimulated ATPase is located on the coccolith vesicle membrane and a P-type Ca 2ϩ -stimulated ATPase is located on the plasma membrane.

Ocean Acidification Reduces Growth and Calcification in a Marine Dinoflagellate

“…These pumps play a key role in generating H + gradients and membrane voltage, which drive multiple transport processes [57], [60]. As indicated from our data, H + -ATPases seem to play an important role in calcification in T. heimii , which is in agreement to observations for E. huxleyi and Pleurochrysis carterae [39], [59], [61]. Here we propose a conceptual model of calcification in T. heimii , which comprises some of the main processes described in this study (Fig.…”

Environmental controls on coccolithophore calcification

“…Measurements of the pH in cytosol, coccolith vesicle and chloroplast of Emiliania huxleyi and Coccolithus pelagicus by Anning et al (1996) showed that the values increase in that order, with the coccolith vesicle 0.2 units higher than the cytosol, but 0.6 to 0.8 units lower than the chloroplast, and 1.1 to 1.2 units lower than the seawater value of pH 8.3. The higher, even by only by a mean of 0.2 units, value of coccolith vesicle than of cytosol pH is difficult to reconcile with the involvement of a V-type H + ATPase pumping H + into the coccolith vesicle (Araki & González 1998, Corstjens et al 2001, Corstjens & González 2004. It must be borne in mind that the mean coccolith vesicle pH in C. pelagicus is relatively higher (7.6−8.3) when cytosol pH is higher than 7.2.…”

“…Half as much ATP is probably needed for the transport of these 3 ions across the coccolith vesicle membrane. In this case, the directly energized process is likely to be Ca 2+ entry using the Ca 2+ ATPase (stoichiometry 2 Ca 2+ influx and probably 2 H + efflux for 1 ATP converted to ADP + P i using a P-ATPase: Evans et al 1991, Anning et al 1996, Araki & González 1998) from the very low free Ca 2+ concentration in the cytosol. HCO 3 − entry could be driven with a relatively small, 10 to 20 mV electrical potential difference (lumen positive with respect to cytosol) produced by the active Ca .…”

Cell Biology, Biochemistry and Genomics of Coccolithophore Biomineralization.

“…Our initial investigations on Pleurochrysis showed that the coccolith vesicle shared enzymatic properties with the Golgi apparatus [9][10]. Our initial investigations on Pleurochrysis showed that the coccolith vesicle shared enzymatic properties with the Golgi apparatus [9][10].…”

“…Golgi type) ATPase is located on the isolated coccolith vesicle [11]. Reproduced from [10] with permission of the Journal of Phycology. Furthermore, we also sequenced the membrane-spanning subunit c of the coccolithophore (P. carterae) V-ATPase to show that the gene encodes a highly conserved protein apparently inserted into the membrane in the manner of all eukaryotic protonpumping ATPases [11], thus strongly suggesting that this proton pump is likely involved in the removal of protons, generated by HCO 3 dehydration, from the coccolith vesicle [12].…”