Discovered over 60 years ago, Rhesus (Rh) proteins are best known as antigens on red blood cells that cause immune reactions during blood transfusions and hemolytic disease in newborn babies. Studies over the past few years have shown that Rh proteins are not limited to red blood cells or to mammals and are involved in the active transport of the ammonium ion(NH4+) or the NH4+ analog,methylammonium, across cell membranes. Most recently, it has been suggested that these proteins are in fact bidirectional channels for biological gases,such as ammonia (NH3) or carbon dioxide (CO2). In support of this hypothesis, CO2 and NH3 exposure causes an increase in Rh1 gene expression in the green alga Chlamydomonas reinhardtii. In order to follow up on this theory, E. Soupene, W. Inwood and S. Kustu decided to eliminate Rh1 function in C. reinhardtii using a technique called RNA interference (RNAi) and found that the protein functions as a CO2 gas channel.
RNAi is a method that silences gene expression by the introduction of double-stranded RNA (dsRNA) specific for that gene; in the case of Soupene's study, the Rh1 gene. Essentially, the team introduced dsRNA that bound to the naturally occurring Rh1 mRNA, which ultimately caused rapid degradation of the Rh1 mRNA and consequently reduced (or even silenced) Rh1 expression. First, the team verified the expression of Rh1 mRNA in C. reinhardtii through northern blot analysis and found that Rh1 expression increased substantially when the green alga was exposed to high environmental CO2. However, in strains of C. reinhardtii that underwent RNAi, the upregulation of Rh1expression seen in response to high environmental CO2 was eliminated. In addition, the alga failed to produce Rh1 protein. Through the use of RNAi, Soupene, Inwood and Kustu have successfully developed a strain of C. reinhardtii that lacks Rh1.
So, how did this new C. reinhardtii strain do without the Rh1 protein? The group determined that Rh1 is required for optimal growth at high environmental CO2 and that a lack of Rh1 protein causes growth defects, in addition to affecting the expression of other genes that normally respond to environmental CO2. The team deduced that algae without Rh1 were unable to rapidly equilibrate with the high CO2environment and, unlike algae with Rh1, did not benefit from the readily available CO2, suggesting that Rh1 functions as a CO2channel. In addition, the group found that strains without Rh1 were able to transport the NH4+ analog methylammonium, suggesting that Rh1 is not involved in the transport of methylammonium, or potentially NH4+, in C. reinhardtii.
Soupene and colleagues have outlined evidence suggesting a possible physiological role for Rh1 as a biological gas channel, which would allow the effective uptake of CO2 and permit optimal growth of C. reinhardtii. While Rh1's role in vertebrates is most likely in waste disposal rather than nutrient uptake, this study, as well as other studies from this group of workers, has given insight into possible alternative physiological roles for Rh proteins.