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

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-C_i cells) or in ambient air (350 μL CO₂ · L−1}; low-C_i 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-C_i 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-C_i 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 C_i cells. The corresponding values for low-C_i 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-C_i cells for the CO_i-concentrating mechanism of C. reinhardtii are discussed.