Lack of the Rhesus protein Rh1 impairs growth of the green alga Chlamydomonas reinhardtii at high CO ₂

Abstract

Although Rhesus (Rh) proteins are best known as antigens on human red blood cells, they are not restricted to red cells or to mammals, and hence their primary biochemical functions can be studied in more tractable organisms. We previously established that the Rh1 protein of the green alga Chlamydomonas reinhardtii is highly expressed in cultures bubbled with air containing high CO 2 (3%), conditions under which Chlamydomonas grows rapidly. By RNA interference, we have now obtained Chlamydomonas rh mutants (epigenetic), which are among the first in nonhuman cells. These mutants have essentially no mRNA or protein for RH1 and grow slowly at high CO 2, apparently because they fail to equilibrate this gas rapidly. They grow as well as their parental strain in air and on acetate plus air. However, during growth on acetate, rh1 mutants fail to express three proteins that are known to be down-regulated by high CO 2: periplasmic and mitochondrial carbonic anhydrases and a chloroplast envelope protein. This effect is parsimoniously rationalized if the small amounts of Rh1 protein present in acetate-grown cells of the parental strain facilitate leakage of CO 2 generated internally. Together, these results support our hypothesis that the Rh1 protein is a bidirectional channel for the gas CO 2. Our previous studies in a variety of organisms indicate that the only other members of the Rh superfamily, the ammonium͞methylammonium transport proteins, are bidirectional channels for the gas NH 3. Physiologically, both types of gas channels can apparently function in acquisition of nutrients and͞or waste disposal.T he Rhesus (Rh) blood group substance, one of the most abundant proteins in red cell membranes, was discovered over six decades ago (1). It is composed of the two antigenic Rh30 proteins and the Rh-associated glycoprotein, RhAG (2-9), which is most closely related to the ancestor of all other Rh proteins (ref. 8 and J. Peng and C.-H. Huang, personal communication). The biochemical function of Rh proteins, which are predicted to have 12 transmembrane-spanning segments, remains controversial (10-12). Human RhAG was reported to be an ammonium import͞methylammonium export system when expressed in Saccharomyces cerevisiae (13) and an ammonium-(methylammonium)͞proton exchanger in oocytes injected with RhAG cRNA (14). Moreover, Rh null red blood cells were found to accumulate more of the ammonium analogue [ 14 C]methylammonium than normal red cells and to lose it less rapidly after being preloaded, leading to the proposal that the Rh blood group substance was an ammonium(methylammonium) export system (15). Although inconsistent, all of the studies cited above concluded that Rh proteins, like their only known paralogues, the ammonium͞methylammonium transport (Amt) proteins [also called methylammonium permeases (Mep) in Saccharomyces cerevisiae], were active transport systems for the ion NH 4 ϩ . Disruption of an Rh gene in the slime mold Dictyostelium discoideum yielded no phenotype (16).Contrary to views of others, we have prop...

Function of human Rh based on structure of RhCG at 2.1 Å

“…Such a role was suggested based on reduced growth of green algae following sRNAi down regulation of a native Rh homolog (34,35). Support for this proposal was augmented by a feature of the prokaryotic NeRh structure (16), where a CO 2 binding site was identified in a cleft near the cytosolic aperture of the channel.…”

Human Rhesus B and Rhesus C glycoproteins: properties of facilitated ammonium transport in recombinant kidney cells

“…However, based on the relatively restricted organ and tissue distribution of Rh proteins and the observation that mutants of Chlamydomonas reinhardtii lacking the paralogue Rh1 protein grow very slowly under high CO 2 conditions, these authors further suggested that mammalian Rh proteins might function as CO 2 rather than as NH 3 gas channels [42,43]. Although the narrowness of the channel in AmtB and the nature of the porelining amino acids [30,31] do not favour the hypothesis of a CO 2 transporter, we cannot formally rule out that Rh glycoproteins are gas channels that might facilitate either NH 3 or CO 2 transport depending on which cell types they are expressed in.…”

Human Rhesus-associated glycoprotein mediates facilitated transport of NH 3 into red blood cells

“…However, it was also proposed recently that the Rh1 protein paralog of the green alga Chla- mydomonas reinhardtii might be regarded as a gas channel for CO 2 , because mutants lacking this protein grow only very slowly when cultured in high CO 2 conditions (48). That Rh proteins might transport NH 3 and͞or CO 2 is a possibility that should be further explored (32).…”