WEIHRAUCH, D.*; MARINI, A.M.; TOWLE, D.W.: Cloning and Expression of a Putative Rh-Like Ammonium Transporter from Gills of the Shore Crab Carcinus maenas

The Rh blood group polypeptides of mammals are related to ammonium transporters in yeasts and plants. RhAG of red blood cells and a kidney homologue RhGK were recently shown to restore ammonium utilization and export in transporter-deficient yeast (Marini et al., Nature 26: 341-344, 2000). The possibility that ammonium transport in gills of the ammonotelic shore crab Carcinus maenas might be mediated by an Rh-like protein was explored using a molecular approach. Poly-A mRNA in gill RNA extracts was reverse transcribed using an oligo-dT primer. Rh-like cDNA sequences were amplified by PCR with degenerate primers. Direct sequencing produced a 1,966-nucleotide cDNA (Accession No. AF364404) coding for a 478-amino-acid protein 39 and 40% identical to human RhAG and RhGK respectively. The putative Rh-like ammonium transporter from C. maenas (RhCM) is 28-43% identical to Rh-like proteins from slime mold, fruit fly, sponge, frog, and mouse. Its level of mRNA expression determined by quantitative RT-PCR was strongest in anterior gills, slightly less strong in posterior gills, and low in antennal glands and hepatopancreas. These expression data paralleled the rates of active ammonia excretion observed earlier in isolated gills. Exposure of crabs to dilute seawater (10 ppt S) containing low (30-50 μmol l-1) or high (300-500 μmol l-1) external ammonia produced no apparent differences in the expression of RhCM mRNA. Experiments are underway to determine the possible ammonium transporting function of RhCM in mutant yeast. Supported by NSF IBN-9807539.