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Nutrient uptake by marine invertebrates: cloning and functional analysis of amino acid transporter genes in developing sea urchins (Strongylocentrotus purpuratus).

Introduction

The transport of amino acids into animal cells is important for many metabolic processes including nutrition, osmoregulation, and protein synthesis (Christensen, 1990). Concentrative transport of amino acids is driven by the energy stored in electrochemical gradients, typically through co-transport with sodium ions (Saier, 2000). Physiologists have described a number of sodium-dependent neutral amino acid transport systems in mammals, including systems A (Christensen et al., 1965), ASC (Christensen et al., 1967), and B (Stevens et al., 1984). Many transport systems take up a broad range of amino acid substrates (Stevens et al., 1982), with certain systems transporting overlapping sets of substrates (Palacin et al., 1998). These studies have shown that cells can transport a given amino acid substrate (e.g., alanine) through multiple transport systems (e.g., systems A, ASC, and B).

The genes that underlie amino acid transport systems like the well-described mammalian systems have been cloned and characterized in a wide range of organisms (e.g., Malandro and Kilberg, 1996; Nelissen et al., 1997; Jack et al., 2000). Cloning these transporter genes has made it possible to obtain experimental proof of function using expression systems including oocytes of the frog Xenopus laevis (Gurdon and Wickens, 1983). Molecular cloning of transporter genes has provided tools for investigating the regulation of transporter gene expression (Santalucia et al., 1992; Lescale-Matys et al., 1993; Corey et al., 1994; Hirsch et al., 1996). Comparisons of protein sequences and physiological functions have made it possible to characterize the phylogenetic relationships among amino acid transporter genes (Saier, 2000; Boudko et al., 2005).

In contrast to the more detailed understanding of genes that regulate amino acid transport systems in model systems, far less is known about the corresponding genes involved in amino acid transport by marine invertebrates. The uptake of dissolved organic material from seawater by marine animals has been investigated for nearly a century (Putter, 1909; Krogh, 1931; Stephens and Schinske, 1961; Jorgensen, 1976; Wright, 1988a; Manahan, 1990; Gomme, 2001). These previous studies have shown that the uptake of dissolved organic material from dilute concentrations in seawater (sub-micromolar) is a physiological process that is broadly distributed among many marine animals, representing most marine phyla. The process is thought to fill osmo-regulatory and nutritional roles, although the quantitative extent of these contributions remains uncertain (Gomme, 2001). Little is known about the specific molecular biological mechanisms involved in these transport processes because very few of the genes associated with amino acid transport by marine animals have been identified (Hosoi, 2005; Toyohara et al., 2005).

Sea urchin embryos and larvae have been used for decades in studies of evolutionary, developmental, and molecular biology (Davidson, 1986; Raff. 1996). Recently, the genome of the sea urchin Strongylocentrotus purpuratus (Stimpson 1857) was sequenced (Sea Urchin Genome Sequencing Consortium et al., 2006). providing a unique evolutionary context for the study of transport processes, since the sea urchin is the first non-chordate deuterostome with a fully sequenced genome (Davidson, 2006). The uptake of dissolved amino acids from dilute concentrations in seawater by developing sea urchins has been characterized in detail (Tyler et al., 1966; Gross and Fry, 1966; Epel, 1972; Manahan et al., 1989). In S. purpuratus, high-affinity amino acid transport systems are active throughout embryogenesis and larval development (Epel, 1972; Manahan et al., 1989). These transport systems function in a sodium-dependent manner (Epel, 1972; Davis et al., 1985) similar to the amino acid transport systems described in mammals (Malandro and Kilberg, 1996). Transport systems with broad substrate specificity (i.e., multiple amino acids transported through a single transport system) have been described in sea urchin embryos (Tyler et al., 1966). These amino acid transport systems in echinoderms are comparable in substrate specificity to some mammalian transport systems--for example. ATB0+ (Epel, 1972; Allemand et al., 1984, 1985). Echinoderm larvae can transport amino acids from dilute solution in seawater at rates sufficient to contribute substantially to the energy requirements of development (Manahan et al., 1983; Shilling and Manahan, 1994). Despite the many detailed studies of amino acid transport systems in developing echinoderms, the genes responsible for regulating flux rates have yet to be identified.

The current study presents (i) the first functional characterization of amino acid transporter genes in a marine invertebrate larval form, (ii) a classification of these genes as members of an evolutionarily conserved gene family, and (iii) a quantitative analysis of the expression of these genes during early sea urchin development. The findings presented assign physiological functions to previously uncharacterized genes that were sequenced during the recently completed sea urchin genome project (Sea Urchin Genome Sequencing Consortium et al., 2006). Additionally, this study highlights the evolutionary significance of the fact that a single transporter gene family can produce diverse physiological functions depending upon context (e.g., expression in different tissues, species, or developmental stages).

Materials and Methods

Cloning and sequencing of amino acid transporter genes

Putative transporter genes were isolated from a cDNA library, representing more than one million clones, that was constructed using purified mRNA from 4-cell-stage embryos of the sea urchin Strongylocentrotus purpuratus ([approximately equal to]4-h-old). Appropriate clones were selected using (32) P-radiolabeled degenerate probes based on conserved regions within previously characterized mammalian transporter genes. The consensus amino acid sequences of these conserved regions were (1) FPYLCYKNGGGAFLIPYFI and (2) GKVVYFTATFP. Nucleotide sequences corresponding to these regions were deduced from codon preference tables for S. purpuratus (Wada et al., 1991). resulting in the following degenerate oligonucleotide probes: (1) TCCCITACCTIT-GCTACAARAACGGHGGIGGHGCHTTCCTIATCCCIT-ACTTCAT; and (2) GGGAAIGTDGCDGTGAAGTAI-ACIACYTTDCC (degenerate nucleotide codes: I = inosine; D = guanine, adenine, or thymine; R = adenine or guanine; and Y = cytosine or thymine). The initial screening of the cDNA library with Probe 1 (corresponding to conserved region 1, above) identified several dozen clones. Further screening with Probe 2 (corresponding to conserved region 2, above), with endonuclease restriction analysis of selected clones, narrowed the range of possibilities to three putative transporter genes, designated as Sp-ATI, Sp-AT2, and Sp-AT3 (S. purpuratus amino acid transporter). Nucleotide sequences for these clones were initially obtained for both DNA strands, using transposon-facilitated insertion of sequencing primers throughout the full length of each cDNA (Strathmann et al., 1991); and cDNA sequences were subsequently confirmed by capillary electrophoresis (Beck-man CEQ: Beckman-Coulter, Fullerton, CA). The nucleotide sequences for Sp-AT1, Sp-AT2, and Sp-AT3 have been deposited in the GenBank database under accession numbers EF538763, EF538764, and EF538765, respectively.

DNA sequence analysis

Structural features of the Sp-AT1, Sp-AT2, and Sp-AT3 transporter proteins were predicted on the basis of the deduced amino acid sequences of the above cDNA clones. Open reading frames (ORFs) of each clone were determined using the Vector NTI software package, ver. 10.3.0 (Informax, Inc., Bethesda, MD). Transmembrane topology models were predicted on the basis of the classical Kyte-Doolittle method (Kyte and Doolittle, 1982) and the "consensus-based" ConPredII method (Arai et al., 2004). Putative O- and N-glycosylation sites were identified using the Center for Biological Sequence Analysis servers (Gupta et al., 2004; Julenius et al., 2005).

The nucleotide sequences of cDNA clones for Sp-ATl. Sp-AT2, and Sp-AT3 were queried against GenBank using TBLASTN (Altschul et al., 1997). These comparisons confirmed that each cDNA clone matches a distinct region in the sea urchin genome. These searches also revealed sequence similarities between the sea urchin genes and previously characterized genes from the SLC6 gene family. Based on these similarities, a phylogenetic analysis was conducted to characterize the position of these sea urchin genes within this well-studied gene family. The protein sequences for a selection of 34 SLC6 genes from human, Caenorhabdites elegans, and Drosophila melanogaster were obtained from National Center for Biotechnology Information (accession numbers are shown in legend for Fig. 3). These genes were aligned with the deduced amino acid sequences of Sp-AT1, Sp-AT2, and Sp-AT3 using ClustalW ver. 1.83 (Thompson et al., 1994). Alignments were minimally adjusted by hand to reduce the number of gaps (Baldauf, 2003), and positions containing gaps were excluded from further analyses using the BioEdit software package, ver. 7.0.9.0 (Hall. 1999). The alignment was bootstrapped (n = 1000) and a consensus neighbor-joining tree based on protein distance was generated using the Phylip sequence analysis package, ver. 3.67 (Felsenstein, 1993). The resulting tree was used to assign each Sp-AT gene to a particular sub-family within the SLC6 gene family.

[FIGURE 3 OMITTED]

Heterologous expression of sea urchin amino acid transporter genes

The sea urchin genes Sp-AT1, Sp-AT2, and Sp-AT3 were cloned into the expression vector pBSTA for expression in frog oocytes. This vector was designed to optimize expression in oocytes of Xenopus laevis (Daudin 1802) by incorporating the 5' and 3' untranslated regions (UTR) of the X. laevis [beta]-globin gene (Goldin, 1991). For Sp-AT1, the entire 1.7-kb cDNA was cloned into the expression vector using BglII. Because of the larger size of Sp-AT2 and Sp-AT3, the UTRs of these genes were excluded and the ORF was cloned into the expression vector. BglII restriction sites were added by PCR amplification with primers containing this site. These primers also introduced a consensus Kozak sequence [A/G]CCATG (Kozak, 1989) at the 5' end of each ORF to improve the translational efficiency in frog oocytes. The primers used to amplify Sp-AT3 were (Fl) 5'-CTCAG-ATCTGCCACCATGCCGTCCAAGGCAAGTTTCTCACC-3' and (R1) 5'-CAGAGATCTCTAGGCAACATTACCAA-CGAC-3'. An internal BglII site in Sp-AT2 was removed by introducing a silent mutation (third nucleotide) into the internal BglII site using a multiple-reaction amplification scheme similar to the strand overlap extension method described by Ho et al. (1989). The primers used for the first PCR reaction were (F2) 5'-CTAGATCTGCCATGCTGT-TCCTGGCCGGTATGCC-3' and (R2) 5'-GAATAGAA-GATTTGAGTTGCTGC-3'. The second reaction used (F3) 5'-GCAGCAACTCAAATCTTCTATTC-3' and (R3) 5'-CGAGATCTTCACGCGTACATCTGTTTGATGGA-3'. The complete ORF for Sp-AT2, now lacking the internal BglII site, was then cloned into pBSTA as described above. The correct DNA sequences for all constructs were verified by restriction digest and capillary sequencing (Beckman CEQ, Beckman Coulter, Fullerton, CA).

In vitro transcription of the linearized cDNA constructs described above was achieved using the T7 mMessage mMachine RNA transcription kit (Ambion. Austin, TX). This resulted in the production of different cRNAs, each containing the ORF of either Sp-AT1, Sp-AT2, or Sp-AT3. Prior to injection into frog oocytes, each cRNA preparation was analyzed on standard denaturing formaldehyde-MOPS agarose gels to confirm molecular weight and integrity (Ausubel et al., 1994).

The cRNA prepared from each of the Sp-AT genes was used for heterologous expression in oocytes of X. laevis according to standard procedures (Quick and Lester, 1994). Individual oocytes were injected with 25 ng (50 nl) of cRNA prepared as described above, or with 50 nl of nuclease-free water as controls. Injected oocytes were incubated for 3-5 days on a rotary shaker (70 rpm) at 18[degrees]C in ND96 + medium (96 mmol [l.sup.-1] NaCl, 2 mmol [l.sup.-1] KC1, 1 mmol [l.sup.-1] Mg[Cl.sub.2], 1.8 mmol [l.sup.-1] Ca[Cl.sub.2], 5 mmol [l.sup.-1] HEPES, 50 [micro]g [ml.sup.-1] gentamycin, pH 7.5). During this culture period, oocytes were examined daily, unhealthy oocytes removed, and the remaining oocytes transferred into fresh medium.

Amino acid transport rates of cRNA-injected oocytes were measured using [[.sup.14]C]alanine as a tracer (Perkin-Elmer, Wellesley. MA) after 3-5 days of incubation with injected cRNA. Transport rates were measured by incubating oocytes in ND96 containing 100 [micro]mol [l.sup.-1] alanine (specific activity, 0.37 kBq nmol [l.sup.-1]; total activity per 200 [micro]1 assay = 187 kBq). Preliminary experiments showed that transport rates of [[.sup.14]C]alanine at a concentration of 100 [micro]mol [l.sup.-1] were linear for over 1 h, so in subsequent experiments oocytes were incubated with [[.sup.14]C]alanine for 40 min. Transport assays were terminated by rinsing each oocyte four times with 10 ml of [Na.sup.+] -free ND96 (96 mmol [l.sup.-1] choline-Cl, 2 mmol [l.sup.-1] KC1, 1 mmol [l.sup.-1] Mg[Cl.sub.2], 1.8 mmol [l.sup.-1] Ca[Cl.sub.2], 5 mmol [l.sup.-1] HEPES, pH 7.5). Oocytes were dissolved with 0.5 ml of 2.5% sodium dodecyl sulfate for 24 h, and the total radioactivity in each sample measured with quench-corrected, liquid scintillation counting. Transport rates were calculated on the basis of the total radioactivity in each oocyte, the specific activity of [[.sup.14]C]alanine in the transport medium, and the elapsed time (pmol Ala [oocyte.sup.-1] [h.sup.-1]). In parallel with the above assays, separate experiments in which [Na.sup.+] -free ND96 was substituted for standard ND96 medium (with [Na.sup.+]) were conducted to evaluate the sodium dependency of the induced amino acid transport activity in frog oocytes from injected sea urchin genes.

Localization of Sp-AT1 expression in larval tissues

To visualize the tissue-specific expression of one of these transporter genes in larvae, antibodies were raised against synthetic peptides designed to match a 20 amino acid residue region of high antigenic index (Jameson and Wolf, 1988) within a predicted extracellular domain of Sp-AT1. This synthetic peptide antigen (RPSEEYWEENVLRQSQSMND) was used to raise rabbit-anti-Sp-AT1 polyclonal antibodies (Bio-Synthesis, Lewisville. TX). Polyclonal antibodies were purified from crude serum by affinity chromatography using the same peptide antigen (Bio-Synthesis, Lewisville, TX).

The specificity of protein binding for this polyclonal antibody preparation was verified by immunoblots of protein (Western blots) extracted from embryos (4-cell-stage) and larvae (4-d-old) of S. purpuratus according to standard methods (Ausubel et al., 1994). Briefly, 10 [micro]g of total protein from each developmental time point was electrophoresed (SDS-PAGE) and transferred to Hybond-ECL nitrocellulose membranes (Amersham Pharmacia, San Francisco, CA). Membranes were blocked with bovine serum albumin (BSA) and incubated with the anti-Sp-AT1 primary antibody at 0.5 [micro]g [ml.sup.-1]. Membranes were washed and incubated with a 1:10,000 dilution of alkaline phosphatase (AP)-conjugated secondary antibody AP-goat-anti-rabbit-IgG (Sigma, St. Louis, MO), and then washed and visualized with Western-Blue AP substrate solution (Promega, Madison, WI).

The tissue-specific expression of the Sp-AT1 amino acid transporter in larvae was assessed by whole-mount antibody staining of pluteus-stage larvae. Larvae were preserved with buffered formalin (4%, pH 7.5). Larvae preserved using the above procedure were rinsed with filtered seawater and incubated in TBST/BSA (20 mmol [l.sup.-1] Tris-HCl, 150 mmol [l.sup.-1] NaCl, 0.05% Tween-20. 0.5% BSA, pH 7.5) for 1 h to block nonspecific antibody binding. Preserved larvae were incubated with the anti-Sp-AT1 primary antibody at 100 [micro]g [ml.sup.-1] for 1 h. Larvae were then rinsed in TBST and incubated for 1 h in TBST/BSA containing the alkaline phosphatase (AP)-conjugated secondary antibody AP-goat-anti-rabbit-IgG (1:10,000 dilution). Larvae were incubated in Western-Blue AP substrate solution (Promega, Madison, WI), and the sites of antibody binding visualized by light microscopy.

Autoradiographic analysis of amino acid transport

Autoradiography was used to identify the tissues involved in transport of dissolved amino acids from seawater by sea urchin larvae. Details of the methods used to expose larvae to radioactive amino acids and prepare sections of larvae for autoradiographic analysis are given in Manahan and Crisp (1983). Briefly, for the current studies pluteus-stage larvae were exposed for 100 min to [[.sup.3]H]alanine added to seawater at a concentration of 1 [micro]mol [l.sup.-1]. Following fixation and dehydration through a graded series of alcohol washes (final being 100% ethanol), larvae were embedded in Spurr's epoxy resin and serially sectioned ([approximately equal to]1 [micro]m thickness). Alternate sections were either stained with Richardson's stain for light microscopy or exposed to autoradiographic emulsion (Kodak Nuclear Track Emulsion) to visualize tissues containing [[.sup.3]H]alanine.

Measurement of alanine transport rates during sea urchin development

Maximum alanine transport capacities were measured throughout early development according to previously published methods (Manahan et al, 1989). Transport rates were measured by suspending embryos or larvae at 1000 individuals [ml.sup.-1] in 0.2-[micro]m (pore size) filtered seawater (FSW) containing 100 [micro]mol [l.sup.-1] [[.sup.14]C]alanine (specific activity, 14 kBq [micro][mol.sup.-1]; total activity, 19 kBq per 10-ml assay). Samples were collected at 2-min intervals and washed on 8.0-[micro]m (pore size) polycarbonate filters using FSW and vacuum filtration. Tissue was solubilized by overnight digestion in 0.5 ml Scintigest (Fisher Scientific, Hampton, NH), and the total activity of each sample was measured by quench-corrected liquid scintillation counting. Transport rates were calculated on the basis of the slopes of regressions of [[.sup.14].C]alanine content per larva against elapsed time. Preliminary measurements using embryos (4-cell-stage) and larvae (4-d-old) from four independent cultures confirmed that transport rates at 100 [micro]mol [l.sup.-1] [[.sup.14]C]alanine were equivalent to those at 200 [micro]mol [l.sup.-1] concentrations. This comparison supports the use of 100 [micro]mol [l.sup.-1] for measuring maximal transport capacities. Alanine transport rates were subsequently measured at this saturating substrate concentration at multiple developmental stages (0-4 d) in an additional four independent cultures of sea urchins.

A separate set of transport rate measurements was conducted to confirm the previously reported broad substrate specificity of amino acid transport systems in developing sea urchins (e.g., Epel, 1972; Allemand et al., 1984) in our experimental material. Pluteus-stage larvae of S. purpuratus (7-d-old) were suspended in FSW containing 50 [micro]mol [l.sup.-1] [[.sup.14]C]alanine alone, or the same concentration of [[.sup.l4]C]alanine with either 500 [micro] mol [l.sup.-1] glycine or 500 [micro]mol [l.sup.-1] serine. Transport assays in the presence or absence of these competing amino acid substrates were conducted essentially as described above.

A separate series of experiments was undertaken to confirm the physiological activity of the Sp-AT1 gene in larvae. The hypothesis tested was that the antibody specific to Sp-AT1 should selectively inhibit alanine transport but not the transport of other non-amino acid substrates by sea urchin larvae (glucose and uridine). These non-amino-acid substrates were selected on the basis that they are known to be transported by different transport systems than amino acids. For these inhibition assays, pluteus-stage larvae (6-d-old) were incubated with and without anti-Sp-ATI antibody (100 [micro]g m[l.sup.-1] in FSW). The rates of transport of alanine, glucose, and uridine in the presence and absence (control) of antibody were conducted as described above. Larvae were enumerated and suspended at 1000 larvae [ml.sup.-1] in FSW with one of three different substrates: [[.sup.14]C]alanine at 5.7 or 19 [micro]mol [l.sup.-1]; [[.sup.14]C]glucose at 3.1 [micro]mol [l.sup.-1]; or [[.sup.3]H]uridine at 0.42 [micro]mol [l.sup.-1]. Transport rates were calculated from the resulting regressions. To assess the effects of anti-Sp-AT1 antibodies on transport rates of alanine, glucose, and uridine, the slopes of regressions obtained from these treatments (presence and absence of antibody) were compared by analysis of variance (ANOVA).

Analysis of Sp-AT1 and Sp-AT2 gene expression during development

Embryos and larvae collected throughout early development (0-4 d) for RNA analysis were homogenized in denaturing solution (4 mol [l.sup.-1] guanidinium thiocyanate, 25 mmol [l.sup.-1] sodium citrate pH 7.0, 0.1 mol [l.sup.-1] [beta]-mercapto-ethanol, and 0.5% sodium sarcosyl) using a rotor-stator homogenizer (Tissue-Tearor, Biospec Products, Bartlesville, OK). Homogenates were immediately frozen by immersion in liquid nitrogen and stored at -80 [degrees]C. RNA was extracted using the RNEasy kit (Qiagen) according to the manufacturer's instructions. RNA was eluted and stored in 1X MOPS/RNasin (40 mmol [l.sup.-1] 3-[n-morpholino]-pro-panesulfonic acid, 10 mmol [l.sup.-1] sodium acetate. 1 mmol [l.sup.-1] EDTA, 100 U [ml.sup.-1] RNasin, pH 7.0).

Quantified RNA samples from each developmental stage sampled were electrophoresed under denaturing conditions (1% agarose, 6% formaldehyde, 1 X MOPS buffer; Ausubel et al., 1994). Preliminary RNA blot analyses of embryos and larvae revealed that Sp-ATI transcripts were more abundant than Sp-AT2. so 5 [micro] of total RNA per sample was used for analysis of Sp-AT1 and 10 [micro]g for Sp-AT2. Gels were stained with ethidium bromide to verify RNA integrity and equal loading. Intensity of ethidium bromide-stained 18S rRNA bands was quantified with the software ImageJ ver. 1.38x (National Institutes of Health) and used to normalize subsequent analyses of [.sup.32]P-labeled band-density in RNA blots (hybridization of [.sup.32]P-labeled DNA probes, details below). RNA was transferred onto nylon membranes (Brightstar-Plus, Ambion, Austin, TX) according to standard downward capillary-transfer methods (Ausubel et al., 1994). Gels and membranes were visualized under UV light to verify complete transfer of RNA. RNA was crosslinked to membranes by exposure to UV light (Stratalinker, Stratagene, La Jolla, CA), and membranes were stored at -80 [degrees]C.

Radiolabeled probes for RNA blot analysis (Northern blots) of Sp-ATI and Sp-AT2 transcript abundance were prepared from cDNA clones. DNA templates for radiolabeling of probes were obtained by restriction digests. The enzymes chosen for Sp-ATI were AatII and PstI resulting in a 889-bp fragment corresponding to a region within the ORF. For Sp-AT2, the enzyme used was NcoI, resulting in a 939-bp fragment corresponding to a region within the ORF. Following restriction digestion, templates were gel purified using the Qiaquick gel extraction kit (Qiagen, Hilden. Germany) according to the manufacturer's instructions. Random-primed radiolabeled DNA probes were then synthesized using the Prime-A-Gene kit (Promega, Madison, WI). To radiolabel each probe, 1.85 MBq of [.sup.32]P-dCTP was used per 50-[micro]l reaction volume (Perkin-Elmer, Wellesley, MA). A molecular-weight marker template (Millennium Marker Template. Ambion, Austin. TX) was also used to generate probes that would hybridize with markers of known molecular weight in each gel. Following synthesis and purification of probes according to the manufacturer's instructions, the radioactivity of each probe was quantified by liquid scintillation counting.

RNA blots were pre-hybridized at 42 [degrees]C for l h in Ultrahyb hybridization solution (Ambion, Austin, TX) according to the manufacturer's instructions. Probes for Sp-ATI and Sp-AT2 were added to achieve final activities of 17 kBq [ml.sup.-1]. Molecular-weight marker probes were added to a final activity of 0.8 kBq [ml.sup.-1]. Hybridizations were conducted overnight at 42 [degrees]C in a rotary hybridization oven (Bambino. Midwest Scientific, St. Louis, MO). Blots were washed, wrapped in plastic, and exposed to Phosphorlmager imaging plates (Amersham Biosciences, Piscataway, NJ) from which digital images were obtained with an FX Molecular Imager (Bio-Rad, Hercules, CA). Measurements of radioactivity (dpm) with Phosphorlmager systems are linear over a wide dynamic range compared to X-ray film (see manufacturer's instructions). Band densities (as pixels) were quantified using ImageJ (NIH) and then normalized to the amount of 18S rRNA to correct for any minor differences in RNA loading among different samples. These mRNA analyses of the expression of Sp-AT1 and Sp-AT2 were performed in duplicate for each developmental stage sampled.

Results

DNA sequence analysis of putative amino acid transporters

Open reading frames (ORFs) were identified within the nucleotide sequences of sea urchin genes Sp-AT1, Sp-AT2, and Sp-AT3. The deduced amino acid sequences are shown in Figure 1. The gene Sp-AT1 contains 164 bp of 5' untranslated region (UTR), a 149-bp 3' UTR, and a 1329-bp ORF encoding a protein calculated to be 48.8 kDa in molecular weight. Sp-AT2 contains a 407-bp 5' UTR, a 447-bp 3' UTR, and an 1857-bp ORF encoding a 69.6-kDa protein. Sp-AT3 contains a 130-bp 5' UTR, an 899-bp 3' UTR, and an 1821-bp ORF encoding a 67.5-kDa protein. The topology models predicted for the Sp-AT genes, based on the classical Kyte-Doolittle hydrophobicity plot (Kyte and Doolittle, 1982) and the consensus-based ConpredII method (Arai et al., 2004), contain multiple transmembrane domains (n = 9-13), intracellular N-termini, extracellular C-termini, and large (68-110 amino acid residues) extracellular domains between transmembrane helices 3 and 4 for all three clones (Fig. 2). A number of putative glycosylation sites were identified on the basis of amino acid sequences (four sites per gene, on average), and the majority of these were located between transmembrane helices 3 and 4. These analyses reveal similarities in sequence and structure between the Sp-AT genes and amino acid transporter genes in other organisms (Saier, 2000).

[FIGURE 2 OMITTED]

Comparison of these Sp-AT cDNA clones with public sequence databases revealed that Sp-AT genes are most similar to the sodium-dependent neurotransmitter transporter gene family (SNF), also referred to as Solute Carrier Family 6 [SLC6] (Wain et al., 2004) or the Sodium: Neurotransmitter/Amino Acid Symporter Gene Family [NSS] (Saier, 2000). We selected the BLAST hit with the best match (lowest e-value) to all three genes (Sp-AT1, Sp-AT2, and Sp-AT3) for multiple sequence alignment: the human glycine transporter gene GLYT2 (accession number: AAD27892). This gene has a 52.7% amino acid sequence identity to Sp-AT1, with 63.8% amino acid sequence similarity (conservative substitutions of a similar amino acid group). Multiple sequence alignment of Sp-AT1, Sp-AT2, and Sp-AT3 with GLYT2 revealed the presence of highly conserved regions, including those for the probes initially designed for isolation clones (horizontal bars in Fig. 1). The deduced amino acid sequences of the different sea urchin transporter genes characterized in the current study were each more similar to previously characterized transporter genes from other species than to the other two sea urchin transporter genes. For example, Sp-AT1 and Sp-AT3 shared 52.9% similarity at the protein sequence level. In contrast, Sp-AT1 shared 63.8% sequence similarity with the human GLYT2 gene, and Sp-AT3 shared 63.4% similarity with the D. melanogaster inebriated gene (accession number: NP_477012). These database searches and sequence alignments support the conclusion that Sp-AT1, Sp-AT2, and Sp-AT3 represent unique transcripts (different genes) present in sea urchin embryos, each of which shares conserved regions with previously characterized transporter genes.

The above sequence comparisons support the annotation of Sp-AT1 Sp-AT2. and Sp-AT3 as members of the SNF gene family. To resolve their position within this family, a phylogenetic analysis was conducted in which these sea urchin genes were compared with 34 other members of the SNF gene family (Fig. 3). This analysis revealed that Sp-ATI is most similar to the amino acid transporter subfamily, a group that includes the human GLYT2 and ATB0+ amino acid transporter genes (accession numbers: NP_004202 and NP_009162, respectively). Bootstrap support for this clade was reasonably high (78%). Sp-AT2 clustered with the C. elegans SNF6 gene (accession number: NP_497416) with weak (50%) bootstrap support. Sp-AT3 was most similar to the D. melanogaster inebriated gene (accession number: NP_477012) with 100% bootstrap support for this node. These analyses confirm the close relationship between the Sp-AT genes and the SNF gene family that was suggested by BLAST searches, and highlight the sequence diversity among the genes identified in the current study.

Testing of physiological function by heterologous gene expression

Significant differences in alanine transport rates were observed between oocytes of Xenopus laevis injected with cRNA encoding either Sp-AT1, Sp-AT2, or Sp-AT3, and control oocytes injected with nuclease-free water (Fig. 4). Oocytes injected with Sp-AT1, Sp-AT2, and Sp-AT3 showed alanine transport rates (given as values plus or minus the standard error) of 570 [+ or -] 23, 603 [+ or -] 28, and 642 [+ or -] 23 pmol alanine [oocyte.sup.-1] [h.sup.-1], respectively, while the corresponding nuclease-free-water-injected controls showed transport rates of only 14 [+ or -] 1.5 pmol alanine [oocyte.sup.-1] [h.sup.-1]. These results demonstrate a greater than 40-fold increase in alanine uptake rates of cRNA-injected oocytes relative to those measured in controls (Student's one-tailed t-test; P < 0.001 for each comparison between cRNA-injected and control oocytes). Oocytes injected with a partial Sp-AT2 construct (deletion of a 51-bp region from the 5' end of the ORF) transported alanine at rates that were not significantly different from those of water-injected controls (t-test; P > 0.05), supporting the conclusion that the transport activity induced by injection of full-length cDNA constructs is a specific property of the genes injected.

[FIGURE 4 OMITTED]

A series of separate experiments investigated the effects of sodium on transport activity of the three genes (Sp-AT1, Sp-AT2, and Sp-AT3). For each gene, transport activity was compared in the presence and absence of sodium, using the same batch of oocytes. For oocytes injected with Sp-AT1 cRNA, alanine transport was significantly greater in the presence of sodium than that of water-injected controls (Student's one-tailed t-test; P < 0.001; n = 5 for both treatments), but there was no difference in transport rates in the absence of sodium (t-test; P > 0.05; n = 5 for both treatments). Similarly, for Sp-AT2, alanine transport rates were significantly greater in cRNA-injected than in water-injected oocytes (t-test; P < 0.05; n = 5 for both treatments). Again, there were no differences in alanine transport in the absence of sodium (t-test; P > 0.05; n = 5). Oocytes injected with Sp-AT3 showed the same pattern, where significant differences in transport rates were observed between control and cRNA-injected oocytes in the presence of sodium (t-test: P < 0.01; n = 7 for control oocytes and n = 9 for cRNA-injected oocytes), but not in the absence of sodium (t-test: P > 0.05; n = 3 for control oocytes and n = 10 for cRNA-injected oocytes). The finding that Sp-ATl, Sp-AT2, and Sp-AT3 mediate alanine transport provides support for annotation of these genes as amino acid transporters.

Localization of Sp-AT1 expression in larval tissues

The major morphological features of the larval stage studied can be seen in the light photomicrograph of a whole-mount pluteus-stage larva (Fig. 5A). In the larval section shown in Figure 5B, the general tissue staining reagent (Richardson's) allows visualization of the same morphological features in a sectioned larva. An autoradio-gram of the location of [.sup.3]H]alanine transported from sea-water by larvae is shown in Figure 5C. In this autoradio-gram both oral and aboral ectoderm show heavy labeling, visualized as white silver grains under dark-field illumination. Exposure of larvae to the anti-Sp-ATl antibody added to seawater revealed specific binding of this antibody to oral and aboral ectoderm (Fig. 5D, E). Figure 5D shows the control measurement of larvae that were exposed to only the secondary antibody and visualized by the presence of alkaline phosphatase. As expected, the endogenous alkaline phosphatase in the digestive system of larvae yielded a positive reaction (dark purple staining in stomach, 'St', seen as darkened region in black-and-white photomicrograph) to the substrate used to visualize the secondary antibody. Figure 5E shows larvae that were exposed to the primary antibody (anti-Sp-AT1) and subsequently reacted with the secondary antibody. The location of the amino acid transporter proteins are seen as the appearance of the dark purple staining of alkaline phosphatase in oral and aboral ectoderm of the whole larvae (Fig. 5E, seen as darkened region in black-and-white photomicrograph). These antibody labeling experiments show that Sp-ATI gene product is located in the ectoderm of sea urchin larvae, which autoradiography data indicate are the same tissues involved in transport.

[FIGURE 5 OMITTED]

Testing of transporter function in larvae by inhibition with Sp-AT1 antibody

The anti-Sp-AT1 antibody was used to test the physiological activity of the transporter protein in sea urchin larvae. Significant differences in alanine transport rates at 19 [micro]mol [l.sup.-1] were measured in the presence and absence of anti-Sp-ATI antibody (Fig. 6A). The rates calculated from the slopes of these regressions differed between larvae in the presence and absence of antibody: 0.71 [+ or -] 0.083 and 1.39 [+ or -] 0.037 pmol alanine [larva.sup.-1] [h.sup.-1], respectively (regressions compared by ANOVA; P < 0.001). Comparison of the slopes of these two regressions demonstrated 49% inhibition of alanine transport rates at the concentration of antibody tested. Further experiments tested the specific inhibition of the Sp-AT1 transporter using the anti-Sp-AT1 antibody. The possibility that nonspecific binding of the antibody might result in inhibition of general transporter properties in ectoderm was tested using non-amino acid substrates. Similar transport inhibition experiments using anti-Sp-ATI antibody on a different batch of larvae were conducted to assess possible inhibition of glucose and uridine transport. In these experiments, the presence of the antibody inhibited alanine transport rates at 5.7 [micro]mol [l.sup.-1] by 40% (regressions compared by ANOVA; P < 0.05). whereas glucose transport rates from 3.1 [micro]mol [l.sup.-1] were equivalent in the presence and absence of the anti-Sp-AT1 antibody, at 0.20 [+ or -] 0.017 and 0.19 [+ or -] 0.021 pmol glucose [larva.sup.-1] [h.sup.-1], respectively. In another set of experiments, the presence of antibody inhibited alanine transport rates by 72% (at 5.7 [micro]mol [l.sup.-1]; regressions compared by ANOVA; P < 0.05). Uridine transport rate was not reduced (with antibody = 0.9 [+ or -] 0.1; without antibody = 1.1 [+ or -] 0.1 fmol uridine [larva.sup.-1] [h.sup.-1], regressions compared by ANOVA; P > 0.05). To confirm the specific binding of the antibody used in these experiments, immunoblots were conducted using 10 [micro]g of protein extracted from embryos (5 h) and larvae (4 d) (Fig. 6B). These experiments revealed a single reactive band in protein preparations from both developmental stages tested, consistent with the specific binding of this antibody to the Sp-AT1 gene product. The single band observed in these blots showed a molecular weight ([approximately equal to]66 kDa) slightly higher than that predicted from deduced amino acid sequence (49 kDa), an observation that could be accounted for by glycosylation of the Sp-ATl protein. These inhibition of in vivo alanine transport rates by this anti-Sp-ATl antibody supports the conclusion that this gene is capable of mediating alanine transport in sea urchin larvae.

[FIGURE 6 OMITTED]

Changes in alanine transport capacities during sea urchin development

The substrate specificity of neutral amino acid transport systems in sea urchin larvae was investigated by measuring the inhibition of alanine transport in the presence of other neutral amino acids present in molar excess. Alanine transport rates were significantly inhibited in the presence of either excess glycine or excess serine (Fig. 7A). Larvae transported [[.sup.14] C]alanine from a 50 [micro]mol [l.sup.-1] solution at a rate of 7.68 [+ or -] 0.68 pmol alanine [larva.sup.-1] [h.sup.-1]. In the presence of 10-fold excess glycine (500 [micro]mol [l.sup.-1]), the [[.sup.14]C]alanine transport rate at 50 [micro]mol [l.sup.-1] was reduced by 89%, to 0.82 [+ or -] 0.1 pmol alanine [larva.sup.-1] [h.sup.-1] (comparison of regressions by ANOVA; P < 0.001). Likewise, in the presence of 500 [micro]mol [l.sup.-1] serine, [[.sup.14]C]alanine transport rate was also reduced by 89%, to 0.86 [+ or -] 0.07 pmol alanine [larva.sup.-1] [h.sup.-1] (comparison of regressions by ANOVA; P < 0.001). These results demonstrate the broad substrate specificity of neutral amino acid transport systems in larvae of S. purpuratus, in which neutral amino acids are competitively transported by the same transport system(s).

[FIGURE 7 OMITTED]

To verify that the transport assay conditions used in these experiments reflect maximal transport capacities ([J.sub.max]), transport rates were measured for early 4- to 8-cell-stage embryos (5 h) and fully formed pluteus-stage larvae (4 d). Previous kinetic analyses showed that maximum transport capacity of alanine by larvae of S. purpuratus occurred at substrate concentrations of 100-200 [micro]mol [l.sup.-1] (Manahan et al., 1989). For the present study, maximum transport capacities for both embryonic and larval stages needed to be defined for comparison of physiological rates to gene expression patterns. Alanine transport rates measured at 100 [micro]mol [l.sup.-1] and 200 [micro]mol [l.sup.-1] in embryos were 1.64 [+ or -] 0.29 and 1.53 [+ or -] 0.08 pmol alanine [embryo.sup.-1] [h.sup.-1], respectively (Fig. 7A). By oneway ANOVA, these rates of transport at different substrate concentrations were not significantly different (df = 1, 4; P > 0.05). Similarly, transport rates measured in larvae were not significantly different between substrate concentrations of 100 and 200 [micro]mol [l.sup.-1] (10.14 [+ or -] 0.11 and 9.14 [+ or -] 1.58 pmol alanine [larva.sup.-1] [h.sup.-1]; ANOVA: df = 1, 4; P > 0.05). These analyses reveal that the average maximal capacities for alanine transport increased 6-fold during development of sea urchin larvae, from an average of 1.6 pmol alanine [embryo.sup.-1] [h.sup.-1] to 9.7 pmol alanine [larva.sup.-1] [h.sup.-1] (Fig. 7B).

Having shown that 100 [micro]mol [l.sup.-1] alanine was sufficient to produce maximum transport rates, a more detailed analysis was undertaken to define changes in maximum transport capacities at 100 [micro]mol [l.sup.-1] throughout embryonic and larval development (Fig. 7C). This developmental increase in transport capacity was described by the equation: transport rate (pmol alanine [individual.sup.-1] [h.sup.-1]) = 2.08*Age + 1.61 (ANOVA of linear regression: [R.sup.2] = 0.96, P < 0.001). The slope of this regression provides a quantitative measure of the increase in maximum alanine transport rates during development of 2.08 [+or -]0.11 pmol alanine [individual.sup.-1] [h.sup.-1] per day of development. As is evident from Figure 7C, maximum transport rates obtained from this series of measurements are highly reproducible between the seven independent cultures tested and represent maximum alanine transport rates for early developmental stages of S. purpuratus.

Ontogenetic changes in Sp-AT1 and Sp-AT2 expression

For comparison of the developmental change in physiological transport rates and levels of transporter gene expression, RNA was extracted from the same cultures of embryos and larvae for which the transport rates are shown as filled symbols (black squares) in Figure 7B. RNA blot analysis (Northern blot) of these samples revealed that the [.sup.32]P-labeled probe for Sp-AT1 hybridized specifically to a single transcript of 5.3 kb in molecular weight (Fig. 8A). The density of this band decreased during embryonic development to the pluteus-stage larvae (0-4 d). Band densities were quantified and normalized to the amount of 18S rRNA (Fig. 8B). These analyses revealed that maximal expression was found in unfertilized eggs, followed by a rapid decline to 57% of this initial abundance within the first 5 h of development (Fig. 8C). The abundance of Sp-AT1 transcript remained relatively constant throughout the remaining period of early development to the 4-d-old larval stage.

[FIGURE 8 OMITTED]

Probes based on Sp-AT2 hybridized specifically to a single transcript of 9.2-kb molecular weight (Fig. 9A). Band densities of radioactivity were quantified and normalized to the amount of 18S rRNA (Fig. 9B). The developmental changes in transcript abundance for Sp-AT2 (Fig. 9C) revealed a pattern different from that of Sp-AT1 (Fig. 8C). Transcript abundance for Sp-AT2 was initially low in unfertilized eggs and subsequently increased 11-fold in larvae (4-d-old). These analyses demonstrate that the amino acid transporter genes Sp-AT1 and Sp-AT2 are expressed throughout early development of sea urchins, but that the developmental patterns of their expression differed markedly, with Sp-AT1 remaining nearly constant while Sp-AT2 increased by an order of magnitude.

[FIGURE 9 OMITTED]

Discussion

Many species of marine invertebrates are capable of obtaining energetically meaningful amounts of nutrition from dissolved organic matter present at low concentrations in seawater (Stephens, 1988; Wright, 1988a; Manahan, 1990; Gomme, 2001). For example, Shilling and Manahan (1994) calculated from transport rates measured in echinoderm larvae that about one-third of the larval metabolic rate could be fueled through the transport of dissolved amino acids from ambient concentrations as low as 50 nmol [l.sup.-1]. The exact contribution of dissolved organic nutrients to the energy budgets of the diverse range of marine invertebrate species is uncertain, but transport rates measured at low ambient concentrations suggest that certain substrates could contribute significantly to energy metabolism (Shilling and Manahan, 1994). Although physiological properties of amino acid transport systems have been described in detail for different developmental stages of echinoids (Tyler et al, 1966; Epel, 1972; Davis et al, 1985, 1988; Manahan et al., 1989), the genes associated with these physiological processes have not previously been identified.

Sequence analyses revealed similarities between the cDNA clones characterized in this study (Sp-AT1, Sp-AT2, and Sp-AT3) and transporter genes previously described in other species. Public database searches revealed similarities with the sodium-dependent neurotransmitter transporter gene family SNF (also known as Solute Carrier Family 6, or SLC6). Phylogenetic analyses have revealed at least six subfamilies within this gene family, including clades specific for [gamma]-aminobutyric acid, monoamine neurotransmitters, or amino acids, as well as other clades specific to certain taxa (e.g., insects or C. elegans) (Chen et al., 2004; Boudko et al, 2005; Thimgan et al, 2006). Protein distance analyses comparing Sp-AT1, Sp-AT2, and Sp-AT3 with a representative sampling of SNF genes reveals the close relationship between these sea urchin genes and those previously characterized SLC6 sequences (Fig. 3). These sea urchin genes each clustered with a different subfamily in this analysis: Sp-AT1 with the amino acid transporter subfamily that includes GLYT1 and ATB0+ (78% bootstrap support), Sp-AT3 with the arthropod inebriated gene (100% bootstrap support), and Sp-AT2 with the C. elegans gene SNF6, although bootstrap support for this last grouping was equivocal (50%).

The finding of an inebriated ortholog (Sp-AT3; Fig. 3) among these cDNA clones was unexpected since these genes have previously been identified only in arthropods (Boudko et al., 2005). This SNF gene is associated with osmoregulation and ion transport, and is expressed in the central nervous system of insects (Chiu et al., 2000) although its substrate remains unknown and a transport function has not been demonstrated in insects. For the sea urchin inebriated homolog Sp-AT3, our findings demonstrate that this gene is capable of mediating alanine transport when expressed in Xenopus oocytes (Fig. 4). This result does not imply that alanine is the preferred substrate for the gene in vivo, but the finding of a transport function for an inebriated homolog remains noteworthy and suggests that further investigation into echinoderm inebriated genes might help to elucidate the physiological roles of these genes across taxa.

The recent publication of the genome sequence for S. purpuratus (Sea Urchin Genome Sequencing Consortium et al., 2006) prompted a further comparison of the genes characterized in the present study (cDNA) with the coding sequences predicted by computational analysis of the sea urchin genome. The Sp-AT1, Sp-AT2, and Sp-AT3 cDNA clones were each compared with the corresponding genomic region (accession numbers: NW_001347949, NW_001354582, and NW_001469296, respectively). For each genomic region, the Sp-AT gene protein sequence was compared with the coding sequence predicted by each of two methods: Gnomon, the gene prediction method used by the National Center for Biotechnology Information; and the FGENESH + gene prediction model (Softberry Inc., Mount Kisco, NY). The Sp-AT clones matched FGENESH + predictions closely, to within 24 amino acid residues. Larger discrepancies were found between the Sp-AT clones and Gnomon predictions. The Gnomon-predicted protein XP_001179122 is 271 residues longer than Sp-AT1, XP_001177914 is 81 residues longer than Sp-AT2, and XP_001196519 is 24 residues longer than Sp-AT3. These discrepancies, in the context of the functional data presented for the cDNA clones described here, emphasize the need for more accurate prediction and experimental analysis of genes that contribute to physiological processes.

The open reading frames (ORF) predicted for the Sp-AT1, Sp-AT2, and Sp-AT3 cDNA clones ranged from 442 to 618 amino acid residues, similar to the range found across the eight families of amino acid transporter genes expressed in animals (300-710 residues for these families; Saier, 2000). The Sp-AT1 ORF is slightly smaller than the 600-700 residues characteristic for the SNF gene family (Saier, 2000). The predicted transmembrane topologies for Sp-AT1, Sp-AT2, and Sp-AT3 contain 9, 11, and 13 transmembrane helices, respectively (Fig. 2). These predicted topologies differ markedly from the well-conserved pattern of 12 transmembrane helices characteristic of the SNF gene family, although they are consistent with the topologies reported among the eight amino acid transporter gene families expressed across animal phyla (6-15 transmembrane helices: Saier, 2000). Despite these unusual features, the cDNA clones characterized in this study are thought to represent complete, functional ORFs for these genes, because they induce alanine transport when expressed in a null system (Fig. 4) and contain characteristic structural features such as the large extracellular loop between transmembrane helices 3 and 4 (Fig. 2) that is well conserved in this gene family (Chiu et al., 2000).

Gene sequences are frequently used to infer putative functions, but direct tests of these assumptions are needed for understanding the molecular biological bases of specific physiological processes. In this study, different DNA constructs, each containing one of the three sea urchin genes cloned from a cDNA library prepared from embryos of S. purpuratus, were used to test physiological function. This was achieved by heterologous expression of sea urchin transporter genes in frog oocytes. These experiments showed that Sp-AT1, Sp-AT2, and Sp-AT3 mediate alanine transport (Fig. 4). Antibody staining revealed that the transporter protein encoded by Sp-AT1 is expressed in both oral and aboral ectoderm of larvae (Fig. 5E), a finding that matches well with the results of autoradiographic analyses which showed that alanine transported from seawater accumulated in these same ectodermal tissues (Fig. 5C). A further testing of transporter function in larvae was undertaken using the anti-Sp-AT1 antibody to inhibit alanine transport; in the presence of antibody, transport was inhibited by [approximately equal to]50% (Fig. 6). Since the other transporter genes identified in this study (Sp-AT2, Sp-AT3) are also expressed in larvae at this developmental stage, the residual transport activity (i.e., the remaining 50%) from these inhibition experiments could be explained in part by their activity. Further experiments with these antibodies might allow estimation of the quantitative contribution of these different genes to the overall activity. The inhibition data shown here confirm the activity of the Sp-AT1 transporter protein in larvae. The sodium dependency of these sea urchin genes is consistent with the extensive literature showing that integumental amino acid transport systems in echinoids and marine invertebrates, in general, are sodium-dependent (Epel. 1972; Davis et al., 1985; Wright, 1988b; Preston, 1993; Gomme, 2001). The amino acid substrate used in our transport assays was alanine, but this does not imply that alanine is the preferred substrate for these transporter genes in vivo. Alanine was chosen because it is a substrate that is taken up by the well-characterized neutral amino acid transport systems of developing echinoderms (Tyler, 1966) and is commonly used to measure the activity of neutral amino acid transport systems in marine invertebrates (Gomme, 2001). The results presented here do not describe the full range of substrates taken up by these transporters, but demonstrate that the Sp-AT genes can transport the "model" substrate alanine in a sodium-dependent fashion. These findings support the conclusion that Sp-AT1 Sp-AT2, and Sp-AT3 represent components of the sodium-dependent neutral amino acid transport system(s) of developing sea urchins described in previous reports (Epel, 1972; Manahan et al., 1983, 1989).

Three different amino acid transporter genes are expressed during development of the sea urchin S. purpuratus (Fig. 1), highlighting the complexity of the relationship between changes in gene expression and physiological transport rates. Of the three genes cloned from the cDNA library screen, genes Sp-AT1 (Fig. 8) and Sp-AT2 (Fig. 9) were both expressed at levels detectable by analyses of RNA blots. Similar blots for the third amino acid transporter gene (Sp-AT3) cloned showed that this gene is present early in development, but nonspecific hybridization on blots prevented quantitative analysis of changes in its expression. There were differences in the levels of expression of Sp-AT1 and Sp-AT2 during development. Preliminary analyses revealed that a larger amount of total RNA was required to produce a detectable signal for Sp-AT1 than for Sp-AT2. The signal intensity obtained for Sp-AT2 from analysis of 10 [micro]g of RNA was about 17-fold lower than the signal obtained from half that amount of RNA for Sp-AT1, suggesting that Sp-AT1 transcripts are about 30 times more abundant in unfertilized eggs. Sp-AT2 increased in expression during development and by the larval stage (4-d-old) had increased to half the level of Sp-AT1.

The increases in maximal transport rates ([J.sub.max]) measured in the present study (Fig. 7) are in close agreement with those previously reported for early embryos (Epel, 1972) and larvae (Manahan et al., 1989) of S. purpuratus. For example, Manahan et al. (1989) reported a [J.sub.max] value for alanine transport by 4-d-old larvae of 12.3 pmol [larva.sup.-1] [h.sup-1], similar to the value reported in the present study of 10.2 pmol [larva.sup.-1] [h.sup.-1] (Fig. 7). Previous studies also reported developmental changes in transporter affinities ([K.sub.t]) for kinetics of amino acid transport. A high-affinity ([K.sub.t] = 1.4 [micro]mol [l.sup.-1]) system is activated shortly after fertilization (Epel, 1972). At the larval stage, a biphasic kinetic transport system is present that contains both high- and low-affinity transporters for amino acids ([K.sub.t] values of 1 and 132 [micro]mol [l.sup.-1]: Manahan et al., 1989). In this context, it is noteworthy that Sp-AT1 and Sp-AT2 are differentially expressed during development. It is possible that these expression profiles are linked to developmental changes in the kinetics of amino acid transport.

Previous studies in our laboratory on changes in ion transport (sodium pump) activities during development of S. purpuratus showed that increases in activities of [Na.sup.+], [K.sup.+]-ATPase (Leong and Manahan, 1997) lagged behind developmental increases in gene expression (amount of mRNA: Marsh et at, 2000). In the current study, amino acid transport rates similarly increased during development, and multiple genes capable of mediating alanine transport were expressed. During the developmental period investigated in the current study (unfertilized egg to 4-d-old larval stage: Figs. 8, 9), there was a 6-fold increase in alanine transport capacity. This change in physiological transport contrasts with the changes measured in expression of the amino acid transporter genes, where Sp-AT1 decreased by 55% (Fig. 8) and Sp-AT2 increased 11-fold (Fig. 9). This contrast between mRNA abundance and physiological rates of an organic-substrate transporter has been observed in other species. For example, ovine intestinal transport of glucose by the [Na.sup.+]-dependant transporter SGLT1 increased by 60-90-fold upon infusion with glucose, while mRNA levels for this gene increased by only about 2-fold in this same treatment (Lescale-Matys et al., 1993). This comparison suggests that multiple mechanisms at the molecular biological level (e.g., transcriptional, translational, and post-translational regulation) are involved in regulating transport rates. Additionally, our observation that gene expression for amino acid transport changes during sea urchin development (Figs. 8, 9) is consistent with other reports of transport physiology in mammalian embryos (Van Winkle, 2001). During mammalian development, changes in the utilization of metabolic substrates are thought to result from the energetic demands of cell division (e.g., macromolecular synthesis; Martin, 2000). Changes in the expression of other transporter genes for amino acids and sugars also occur during mammalian development (Ito and Groudine, 1997; Pantaleon et al., 2001). Mammalian glucose transporters provide another example, where GLUT1 is expressed at high levels during fetal stages of mouse development, while expression of a second glucose transporter gene (GLUT4) is initiated post-natally (Santalucia et al., 1992).

The finding that multiple amino acid transporter genes are expressed during development of S. purpuratus is supported by preliminary experiments we have undertaken with other species of sea urchins. Partial clones of putative amino acid transporter genes have been obtained from developmental stages of two other urchin species, based on a polymerase chain reaction analysis using oligonucleotide primers designed from the same conserved regions as those used for probe design in the present study (Fig. 1). For embryos of the temperate urchin Lytechinus pictus, three distinct clones were obtained on the basis of deduced amino acid sequences (ranging from 52% to 56% similarity in amino acid sequence between these clones). Comparison of the deduced amino acid sequences of the three cDNA clones from L. pictus with the amino acid transporter genes from S. purpuratus revealed a high sequence similarity for one clone (99% similarity between Sp-AT2 and a clone from L. pictus). In another species, the Antarctic sea urchin Sterechinus neumayeri, two distinct clones were present in embryos (with 45% amino acid sequence similarity between these clones). These preliminary results suggest that expression of multiple amino acid transporter genes during development may be widespread among different sea urchin species. Further experiments using cloned genes should provide novel insights into the evolutionary relationships of amino acid transporters in sea urchins, and the physiological roles of transporter proteins during development in diverse environments.

This identification of amino acid transporter genes in S. purpuratus represents a substantial increase in our understanding of how developmental stages of marine invertebrates transport amino acids from seawater. These findings demonstrate that amino acid transporter genes are expressed in developing sea urchins and that the genes involved are closely related to genes associated with synaptic neurotransmitter transport (SNF family). This relationship with gene families involved in neuronal signaling is worth noting, since Sp-AT1, Sp-AT2, and Sp-AT3 were cloned from early embryonic stages lacking a nervous system (i.e., 4-cell-stage embryos). The SNF gene family serves a number of well-characterized physiological roles in diverse organs and tissues, including the nervous, renal, and digestive systems (Malandro and Kilberg, 1996; Palacin et al., 1998; Saier, 2000). In contrast, the expression of these genes in the ectoderm of developing sea urchins (e.g., Sp-AT1 in Fig. 5D, E) represents a context in which the physiological roles of these genes have not been characterized. In sea urchin larvae, the transporter proteins are located in ectodermal tissues that are in direct contact with seawater, facilitating the transport of organic nutrients from the environment. In these developmental stages of sea urchins, amino acid transport can play an important nutritional role (Manahan, 1990). Osmoregulatory roles for members of this gene family have been shown to be crucial for survival of mammalian embryos (Van Winkle, 2001; Steeves et al., 2003), suggesting this as another possible physiological role during sea urchin development--that is, maintenance of high intracellular concentrations of organic osmolytes (Wright and Secomb, 1986; Gomme, 2001). As a neurotransmitter transporter gene family, SNF genes also play a major role in neural signaling and sensory physiology. Small molecular weight organic compounds dissolved in seawater are known to serve as chemical cues for fertilization, for aspects of larval behavior, and as inducers of metamorphosis (Zimmer-Faust and Tamburri, 1994; Riffel et al., 2002; Hadfield and Koehl, 2004). Some of these chemical cues are amino acids or their derivatives. The expression of SNF transporter genes in sea urchin embryos and larvae, along with receptors yet to be identified, suggests a possible role for the SNF gene family in chemosensory processes in marine organisms.

In conclusion, the findings reported here demonstrate that the sea urchin genes Sp-AT1, Sp-AT2, and Sp-AT3 encode functional amino acid transporters and are expressed during embryonic and larval stages of development. Ontogenetic increases in maximum alanine transport rates correspond to changes in the expression of Sp-AT genes, suggesting that these genes play a role in regulating transport physiology during development. The results presented here constitute, to our knowledge, the first molecular biological and functional characterization of amino acid transporter genes in a larval form of marine invertebrates. The identification of these genes will facilitate further studies into the mechanisms underlying the activation of transport systems at fertilization (Epel, 1972) and known ontogenetic changes in transport kinetics during later larval development (Manahan et al., 1989). These findings constitute an essential step toward understanding the mechanisms underlying the physiological role and regulation of amino acid transport processes during development of marine invertebrates.

Acknowledgments

We are especially grateful to Dr. Eric H. Davidson for providing cDNA libraries and to EHD and Dr. Andy Cameron for their expert guidance in the early stages of this work. Dr. Adam G. Marsh assisted with the antibody staining experiments and some of the transport assays. D. Dimster-Denk assisted with autoradiographic analyses. Amanda Haag helped to produce the gene expression constructs. Dr. Patricia von Dippe and Andrew Nosal assisted with preliminary analysis of transporter genes in other sea urchin species. Dr. Michael Quick provided oocytes and helpful advice with many aspects of the heterologous expression experiments. This research was supported by the National Science Foundation under grant numbers 0130398 and 0412696 to DTM.

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ELI MEYER* AND DONAL T. MANAHAN[dagger]

Department of Biological Sciences, University of Southern California, Los Angeles, California 90089-0371

Received 9 October 2007: accepted 22 April 2009.

* Current address: Department of Integrative Biology. University of Texas at Austin, Austin. TX 78712.

[dagger] To whom correspondence should be addressed. E-mail: manahan@usc.edu
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Author:Meyer, Eli; Manahan, Donal T.
Publication:The Biological Bulletin
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Geographic Code:1USA
Date:Aug 1, 2009
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