Resolving the biological role of the Rhesus (Rh) proteins of red blood cells with the aid of a green alga

The presence or absence of Rh antigens, abundant components of the red blood cell (RBC) plasma membrane, is a major cause of blood transfusion incompatibility and of hemolytic disease of the newborn. As deduced from studies on Rh_null erythrocytes, the Rh complex of RBC includes the Rh-associated glycoprotein RhAG, the Rh polypeptides, CD47, LW proteins, and glycophorin B (1). RhAG is essential for the assembly of the Rh complex, and mutations in RhAG result in the Rh_null syndrome (2). Despite their significance and intensive research, the biological role of the Rh protein complex remains controversial. This controversy may be accounted for by the fact that RhAG homologues are found in other mammalian tissues and in many lower organisms (3), including unicellular eukaryotes (4). Studies by Marini and colleagues (3) identified some homology (20–27% identity) between RhAG and Mep/Amt ammonium and methylammonium transporters in various organisms and suggested that the erythroid RhAG may function as an ammonium transporter (3). This finding was supported by the observation that RhAG (and its kidney-located homologue) could complement a yeast mutant impaired in ammonium uptake. Further, RhAG enhanced efflux of a preloaded methylammonium from yeast, suggesting that it might be involved in ammonium export as well (5). The notion that RhAG and its homologues are involved in transfer gained additional support by the observation that expression of RhCG, a mammalian nonerythroid homologue of RhAG, along the rat nephron, matched that expected from ammonium excretion activities of the respective nephron sections (6). An exciting contribution from the laboratory of S. Kustu (7) in this issue of PNAS provides supporting evidence for their earlier suggestion (4) that RH1 of the green alga Chlamydomonas reinhardtii, which is highly homologous to RhAG, may in fact function as a CO₂ channel. In their earlier study Soupene et al. (4) showed that the expression of rh1 was strongly up-regulated after exposure of Chlamydomonas cells to high (3% vol/vol) levels of CO₂. In the present study (7) the authors used RNA interference technology to lower the abundance of RH1 mRNA. The three lines selected for further analysis did not express rh1 but induced low-CO₂-dependent genes normally when grown in an air level of CO₂ and showed reduced growth under high-CO₂ conditions. Uptake of methylammonium hardly differed between the wild-type and the rh1 RNA interference strains.

RH1 of the green alga Chlamydomonas reinhardtii may function as a CO₂ channel.

Many photosynthetic microorganisms possess inducible mechanisms that concentrate CO₂ at the carboxylation site, compensating for the relatively low affinity of ribulose-1,5-bisphosphate carboxylase/oxygenase for CO₂ and allowing acclimation to a wide range of CO₂ concentrations (see refs. 8–16 and references therein). The organization of the carboxysomes in prokaryotes and the pyrenoids in eukaryotes, and the presence of membrane mechanisms for inorganic carbon (Ci) uptake, are central to the activity of the CO₂-concentrating mechanism. The presence of multiple Ci-transporting systems in cyanobacteria has been indicated (17), but little is known about the mechanism of Ci uptake in eukaryotes, such as the green alga Chlamydomonas (13). As in most other photosynthetic organisms examined to date, Chlamydomonas can use either CO₂ or or both (13). Kinetic data based on initial rates of transport are available for Ci uptake in some organisms but difficult to obtain for Chlamydomonas, because the steadystate internal Ci concentration is attained rapidly. This finding is in agreement with the proposal that CO₂ can cross the membrane quickly by specific channels (7).

Facilitation of CO₂ formation from in the periplasmic space by carbonic anhydrase (CA) (18), encoded by Cah1; within the pyrenoid by thylakoid lumen-located CA (10, 19), encoded by Cah3 (ctCA); and possibly the involvement of a mitochondrial-located CA, encoded by mtCA (20), enhances the supply of CO₂ to ribulose-1,5-bisphosphate carboxylase/oxygenase (Fig. 1). As opposed to CO₂ supply, there is far less information regarding the mechanism of uptake in Chlamydomonas. Nevertheless, isolated chloroplasts of Chlamydomonas can accumulate to levels higher than could be accounted for by passive diffusion (13). Furthermore, Ci uptake is higher in chloroplasts isolated from low- than from high-CO₂-grown cells (8). Genes involved in Ci uptake by Chlamydomonas are strongly induced when high-CO₂-grown cells are transferred to low-CO₂ conditions (12, 13, 19, 20). Mutants in which these genes were impaired required high CO₂ for growth. In contrast, expression of rh1 is induced under high CO₂ and suppressed under low CO₂ (4, 7). The function of RH1 as a CO₂ channel, as suggested, would allow influx of CO₂ when all other (known) means of mediated Ci influx are suppressed (i.e., under high levels of ambient CO₂), whereas the absence of RH1 under low CO₂ would minimize wasteful leakage of CO₂ from the cells. Thus, assignment of RH1 as a CO₂ channel is novel to our understanding of the mechanisms and components involved in CO₂ acquisition by photo-synthetic microorganisms.

Fig. 1.

A simplified scheme of inorganic carbon uptake and accumulation in C. reinhardtii. The putative function of RH1 as a chloroplast envelope-located CO₂ channel is emphasized. The means by which is taken up across the plasmalemma, the chloroplast envelope, and the thylakoid, if any, is not known. Possible involvement of other components, including the mitochondrial-located CA, LIP-36, and pmp-1 (8, 13), is not indicated. For the sake of simplicity, diffusion of CO₂ across the lipid bilayer of the various membranes is not shown. Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase; PGA, 3-phosphoglyceric acid.

Although not yet examined experimentally, the presence of a typical N-terminal transit peptide suggested that RH1 is located within chloroplast envelope (4) (Fig. 1) where the low-CO₂-induced LIP-36 also resides. Should RH1 be confined to the chloroplast envelope, transfer of CO₂ across the plasmalemma of Chlamydomonas may limit Ci uptake under high-CO₂ conditions.

The function of RH1 as a CO₂ channel allows influx of CO₂ when other means are suppressed.

The possibility that CO₂ does not merely diffuse through the bulk lipids within membranes was indicated in experiments where an aquaporin blocker severely inhibited CO₂ uptake by Synechococcus sp. strain PCC 7942 (21). Another means of passage of CO₂ across the membrane was suggested by Forster and colleagues (22) who provided 4,4′-diisothiocyanato-stilbene-2,2′-disulfonate to RBCs. This treatment resulted in reduced CO₂ permeability without affecting the CA activity. 4,4′-Diisothiocyanato-stilbene-2,2′-disulfonate is known to inhibit the exchanger (band 3) in the RBC mem brane. Recent studies by Tanner (23) and Bruce et al. (24) showed that the two major complexes, Rh and the exchanger, are ciated closely assoforming a single macrocomplex that may function as an integrated CO₂/O₂ gas exchange unit in the erythrocyte membrane.

Finally, both RBCs and the soil alga Chlamydomonas must cope with high CO₂ levels in their surroundings and have apparently adopted a similar channel mechanism to allow efficient exhaustion or uptake of CO₂. It is plausible that ability of the alga to acclimate to low CO₂ conditions was acquired simultaneously with development of the means to block the expression of rh.