MANAHAN, D.T.: Molecular physiology of amino acid transport during sea urchin development

Classic studies on eggs of sea urchins have shown that many biological processes are activated at fertilization, such as changes in membrane transporters and increases in protein synthesis. After fertilization the ontogenetic changes that occur in rates of amino acid transport have also been well documented. The mechanisms of activation of amino acid transporters and the molecular biological bases for the subsequent changes in affinities and capacities of such transporters during development are, however, not understood. Putative amino acid transporter genes were cloned from a cDNA library of 4-h-old embryos of the purple sea urchin Strongylocentrotus purpuratus. Oligonucleotide probes were designed from codon preference data for this species and the probes were used to select three cDNA clones following double plaque lifts (based on counter screens of all positives from the first probe with a second probe of different nucleotide sequence). Each of the three full-length clones are distinct in sequence with deduced amino acid sequences of 442, 533, and 607 residues. All three sea urchin clones show amino acid identity to known ion-dependent amino acid transporters in mammals. Antibodies raised against specific regions of the presumed extracellular domains of each sea urchin clone revealed that these proteins are localized in the body-wall epithelium of larvae, where amino acid transport from seawater is known to occur. Transport of neutral amino acids from seawater was inhibited when larvae were pre-incubated in the antibody to the 442 amino acid protein. This suggests that this protein might be a neutral amino acid transporter. The finding of multiple amino acid transporter proteins in sea urchin embryos will facilitate further studies of the regulation of amino acid transport during development.