, Volume 200, Issue 3, pp 358-368

The CO2 permeability of the plasma membrane of Chlamydomonas reinhardtii: mass-spectrometric 18O-exchange measurements from 13C18O2 in suspensions of carbonic anhydrase-loaded plasma-membrane vesicles

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Abstract

The unicellular green alga Chlamydomonas reinhardtii possesses a CO2-concentrating mechanism. In order to measure the CO2 permeability coefficients of the plasma membranes (PMs), carbonic anhydrase (CA) loaded vesicles were isolated from C. reinhardtii grown either in air enriched with 50 mL CO2 · L−1} (high-Ci cells) or in ambient air (350 μL CO2 · L−1}; low-Ci cells). Marker-enzyme measurements indicated less than 1% contamination with thylakoid and mitochondrial membranes, and that more than 90% of the PMs from high and low-Ci cells were orientated right-side-out. The PMs appeared to be sealed as judged from the ability of vesicles to accumulate [14C]acetate along a proton gradient for at least 10 min. Carbonic anhydrase-loaded PMs from high and low-Ci cells of C. reinhardtii were used to measure the exchange of 18O between doubly labelled CO2 (13C18O2) and H2O in stirred suspensions by mass spectrometry. Analysis of the kinetics of the 18O depletion from 13C18O2 in the external medium provides a powerful tool to study CO2 diffusion across the PM to the active site of CA which catalyses 18O exchange only inside the vesicles but not in the external medium (Silverman et al., 1976, J Biol Chem 251: 4428–4435). The activity of CA within loaded PM vesicles was sufficient to speed-up the 18O loss to H2O to 45360–128800 times the uncatalysed rate, depending on the efficiency of CA-loading and PM isolation. From the 18O-depletion kinetics performed at pH 7.3 and 7.8, CO2 permeability coefficients of 0.76 and 1.49·10−3} cm·s−1}, respectively, were calculated for high Ci cells. The corresponding values for low-Ci cells were 1.21 and 1.8·10−3} cm·s−1}. The implications of the similar and rather high CO2 permeability coefficients (low CO2 resistance) in high and low-Ci cells for the COi-concentrating mechanism of C. reinhardtii are discussed.

We are grateful to Prof. H.P. Fock (FB Biolgie, Universität Kaiserslautern, Germany) for providing us with the MS facilities, laboratory equipment and critically reading the manuscript. We wish to thank Prof. T. J. Buckhout (Angewandte Botanik, Humbold Universitat, Berlin, Germany) for his help during isolation of PMs and Drs. A. Tubbe (FB Biologie, Universitat Kaiserslautern, Germany) and D. Kramer (Institut für Botanik der Technischen Hochschule, Darmstadt, Germany) for the measurement of radioactive acetate uptake and support with freeze fracture and electron microscopy, respectively. In addition, we thank Drs. M. R. Badger and S. von Caemerer (Research School of Biological Sciences, The Australian National University, Canberra, Australia) for many helpful comments during preparation of the manuscript. Part of this work was supported by a research fellowship from the Deutsche Forschungsgemeinschaft awarded to D.S.