ISOLATION AND CHARACTERIZATION OF GOLGI FROM COCCOLITHUS PELAGICUS (PRYMNESIOPHYCEAE)¹

Abstract

Cells of the marine alga Coccolithus pelagicus (Wal‐lich)J. Schiller grown in axenic cultures were homogenized and fractionated. The distribution of organelle markers was assessed enzymatically after centrifugation through zonal, density, and flotation gradients made with sucrose, sorbitol, or Percoll. Mitochondria (1.19 g·cm‐3) and chloroplasts (1.15 g·cm‐3) were recovered in sucrose gradients at densities similar to those observed for higher plants and most algae. The position of endoplasmic reticulum and plasma membrane in the gradients was monitored by NADPH cytochrome c reductase and vanadate‐sensitive Mg2+‐ATPase, respectively. Higher plant Golgi markers, latent undine diphosphatase (UDPase) and glucan synthase I, were colocalized at a density range including two peaks of activity at 1.13–1.15 g·cm‐3. Bound calcium was associated with high density (1.15 g·cm‐3) membranes. Ca2+‐stimulated ATPase was found at high levels on membranes that did not coisolate with the latent UDPase‐containing membranes. The Ca2+‐stimulated ATPase, a possible participant during calcification, was associated with a chloroplast‐enriched fraction in all the organelle separation systems. However, about 30% of the total activity was separated from both the chloroplasts and Golgi on 0–70% Percoll gradients containing 0.4 M sucrose. The possible relationship of the Golgi and the high‐density organelle exhibiting Ca2+‐stimulated ATPase to coccolithogenesis and the process of calcification and crystal formation is discussed.

A COCCOLITHOPHORID CALCIFYING VESICLE WITH A VACUOLAR‐TYPE ATPASE PROTON PUMP: CLONING AND IMMUNOLOCALIZATION OF THE V0 SUBUNIT c

“…Cultures of P. carterae (strain 136, Plymouth Marine Biological Laboratory, UK; previously referred to as Hymenomonas carterae; van der Wal et al 1983 and Pleurochrysis sp. strain CCMP299 (Culture Collection of Marine Phytoplankton, Bigelow Laboratories, West Boothbay Harbor, ME) were grown in natural or synthetic seawater with f/2 nutrients at 16–18° C and a 12‐h light:dark cycle as previously described (Wainwright et al 1992, Araki 1997).…”

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

“…strain CCMP299 was obtained from the Center for Culture of Marine Phytoplankton (CCMP) in Maine. Cultures were maintained under axenic conditions as peviously described (Wainwright et al 1992).…”

“…Cell-free homogenate (10 mL) was overlayed on a sucrose density gradient-15% (4 mL)-30% (4 mL), 30% (10 mL)-60% (10 mL)-and centrifuged at 6500 rpm for 20 h (w 2 t ϭ 3.34 ϫ 10 10 ·s Ϫ1 ) in an SW27 rotor and a Beckman L8-M ultracentrifuge. Fractions (0.9 mL) were collected from the top of the tube, as previously described (Wainwright et al 1992). Organelle distributions in the gradients were determined by means of marker enzymes (i.e.…”

“…Okazaki et al (1984) first reported Ca 2ϩ -stimulated ATPase in a coccolithophorid (Cricosphaera roscoffensis) cell extract. We later showed that Ca 2ϩ -stimulated ATPase was present at high levels in a calcifying strain of Pleurochrysis and distributed among all the major membranous fractions (Wainwright et al 1992, Kwon andGonzález 1994). This diverse distribution made it clear that Ca 2ϩ -stimulated ATP hydrolysis could be coupled to a variety of enzymatic functions, depending on actual substrates, membrane localization, and enzyme complex orientation in the respective organellar membrane, not all of which are necessarily directly involved in calcification.…”

Photosynthesis and inorganic carbon utilization in Pleurochrysis sp. (Haptophyta), a coccolithophorid alga

“…A high calcifying (HC) strain of Pleurochrysis sp. (CCMP299, Center for Culture of Marine Phytoplankton, Bigelow Laboratory, Maine) and a low calcifying (LC) Pleurochrysis elongata (CCAP961/3, Culture Collection of Algae and Protozoa, Scotland) were grown axenically at 15OC and pH 7.5 as described previously (Wainwright et al 1992). Cultures used in subsequent experiments were harvested at 5 wk (Quiroga & Gonzalez 1993).…”