WEIHRAUCH, Dirk; MARINI, Anne-Marie; TOWLE, David/W; Uni. of Osnabrueck, Osnabrueck; Universit� Libre de Bruxelles, Bruxelles; MDIBL, ME: Rh-like ammonia transporter in crustaceans: Expression analysis and functional studies of the branchial ammonia transporter RhCM from the shore crab Carcinus maenas.

Like most aquatic living animals, crustaceans excrete their nitrogenous waste products largely as ammonia. Recently we have discovered a Rhesus-like protein in C. maenas gills that shows high homologies to the human Rhesus associated proteins, identified as ammonium transporters. A 1,966-nucleotide cDNA coding for this 478-amino-acid protein was cloned and sequenced employing poly-A mRNA from C. maenas gill (GenBank Acc.: AF364404). From a hydrophobicity blot of the amino acid sequence, twelve transmembrane domains were predicted. Real-time quantitative PCR mRNA expression analysis of various tissues from the shore crab revealed that the putative crustacean ammonium transporter (RhCM) is exclusively expressed in the ammonia excreting gills. These data were confirmed by Western blot analysis employing an RhCM-specific antibody. Expression of RhCM in a yeast mutant lacking endogenous ammonia transporters partly recovered reduced growth when cells where grown on media with ammonia as the single nitrogen source. A result that indentifies RhCM as an functional ammonia transporter. RhCM was also cloned from gills of crustaceans occupying different haline environments, such as Callinectes sapidus (5 ppt), Cancer irroratus (35 ppt), Dilocarcinus pagei (freshwater) and the isopod Idotea baltica (20 ppt). The high degree of conservation with ammonia transporters found in fungi, bacteria and archaebacteria (~ 20%), as well as the striking homology to mammalian ammonia transporters (> 40%) from kidney, lead to the suggestion that proteins of the Rh-family play an universal role in ammonia transport.