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. 2010 Jan;24(1):206–217. doi: 10.1096/fj.09-135889

Caenorhabditis elegans P5B-type ATPase CATP-5 operates in polyamine transport and is crucial for norspermidine-mediated suppression of RNA interference

Alexander Heinick *, Katja Urban *, Stefan Roth *, Danica Spies *, Frank Nunes †, Otto Phanstiel IV ‡, Eva Liebau *, Kai Lüersen *,1
PMCID: PMC2797033  PMID: 19762559

Abstract

Physiological polyamines are required in various biological processes. In the current study, we used norspermidine, a structural analog of the natural polyamine spermidine, to investigate polyamine uptake in the model organism Caenorhabditis elegans. Norspermidine was found to have two remarkable effects: it is toxic for the nematode, without affecting its food, Escherichia coli; and it hampers RNA interference. By characterizing a norspermidine-resistant C. elegans mutant strain that has been isolated in a genetic screen, we demonstrate that both effects, as well as the uptake of a fluorescent polyamine-conjugate, depend on the transporter protein CATP-5, a novel P5B-type ATPase. To our knowledge, CATP-5 represents the first P5-type ATPase that is associated with the plasma membrane, being expressed in the apical membrane of intestinal cells and the excretory cell. Moreover, genetic interaction studies using C. elegans polyamine synthesis mutants indicate that CATP-5 has a function redundant to polyamine synthesis and link reduced polyamine levels to retarded postembryonic development, reduced brood size, shortened life span, and small body size. We suggest that CATP-5 represents a crucial component of the pharmacologically important polyamine transport system, the molecular nature of which has not been identified so far in metazoa.—Heinick, A., Urban, K., Roth, S., Spies, D., Nunes, F., Phanstiel IV, O., Liebau, E., Lüersen, K. Caenorhabditis elegans P5B-type ATPase CATP-5 operates in polyamine transport and is crucial for norspermidine-mediated suppression of RNA interference.

Keywords: S-adenosylmethionine decarboxylase, body size, life span, ornithine decarboxylase, spermidine

The polyamines putrescine, spermidine, and spermine are ubiquitous components of cells that affect numerous biological processes. Most polyamine functions are related to their polycationic character, allowing them to interact with anionic binding sites of macromolecules. In particular, processes where nucleic acids are involved, such as gene transcription and translation, have been shown to be influenced by changing polyamine concentrations. Because adequate polyamine levels are required for eukaryotic cell cycle progression, organisms control their intracellular polyamine pattern precisely by synthesis, catabolism, and transport. Hence, targeting polyamines and their metabolism by polyamine analogues and/or enzyme inhibitors is suggested to be a promising chemotherapeutic strategy for antiproliferative intervention (1,2,3).

In the synthesis pathway, the key enzymes ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) provide putrescine and the aminopropyl moiety donor decarboxylated S-adenosylmethionine, respectively, which are used by spermidine synthase to produce spermidine. A spermine synthase adds a second aminopropyl group, leading to spermine. Usually, higher polyamines can be back-converted or degraded by polyamine oxidase reactions, often preceded by an N-acetylation step (2, 3). Polyamine uptake has been analyzed by biochemical and pharmacological means in many eukaryotic cell types. It was found to be carrier mediated, energy dependent, and Na+ activated (4). Nevertheless, there is only little information on the molecules involved (5), despite the fact that polyamine transporter genes have been identified in several prokaryotes and unicellular eukaryotes (6,7,8).

Caenorhabditis elegans is a simple but powerful model to study general physiological topics in a multicellular organism. Gene functions can be examined by forward and reverse genetic approaches, e.g., applying chemical mutagenesis or RNA interference (RNAi) (9, 10). Moreover, comparative genome analyses revealed a remarkable degree of conservation between biochemical pathways present in C. elegans and mammals (11). Likewise, the nematode possesses a polyamine synthesis with ODC, AdoMetDC, and spermidine synthase. A spermine synthase is lacking. The C. elegans ODC loss-of-function mutant odc-1(pc13) has been previously shown to exhibit only a minor phenotype under standard culture conditions. However, in follow-up analyses, odc-1(pc13) was found to be polyamine auxotroph, since its embryonic development depends on exogenous polyamines (12, 13). These results gave strong evidence for the presence of a polyamine transport system, by which the nematode is able to largely compensate the loss of polyamine synthesis.

Polyamine analogues and polyamine conjugates have been established as promising tools for analyzing polyamine transport (5, 14,15,16,17). In the present study, we have used the toxic spermidine analog norspermidine in a chemistry-to-gene screen to isolate the C. elegans mutant strain nor-2. A deletion in the P-type ATPase CATP-5 has been demonstrated to be responsible for the norspermidine-tolerant phenotype. Genetic interaction studies suggest that CATP-5 has a function redundant to polyamine synthesis and link polyamines to important postembryonic processes of the multicellular organism.

MATERIALS AND METHODS

Oligonucleotides

Oligonucleotide sequences are listed in Supplemental Table 1.

C. elegans culturing, strains, and generation of double mutants

Worms were maintained on nematode growth medium (NGM) at 20°C under standard conditions using Escherichia coli OP50 as a food source (18). Worm populations were synchronized by alkaline hypochlorite lysis (19). The following strains were obtained from the Caenorhabditis Genetics Center at the University of Minnesota (Minneapolis, MN, USA), which is funded by the National Institutes of Health National Center for Research Resources: wild-type N2 Bristol; wild-type CB4856 (Hawaii); SF1 odc-1(pc13::Tc1)V; DA438 bli-4(e937)I, rol-6(e187)II, daf-2(e1368) vab-7(e1562)III, unc-31(e928)IV, dpy-11(e224)V, and lon-2(e678)X; and EM122 stDp2(X;II)/+ II, him-5(e1490)V, and unc-18(e81) dpy-6(e14)X. Strain FX02772 smd-1(tm2772)I was obtained from the National Bioresource Project, Tokyo Women’s Medical University School of Medicine. It was outcrossed 8 times to wild-type N2 Bristol prior to phenotype characterization. The appearance of the odc-1(pc13::Tc1), smd-1(tm2772), and catp-5(mun1) alleles in progeny derived from genetic crosses was verified by PCR. The primer pair ODC-1-S and ODC-1-AS amplifies a 1373-bp fragment for the wild-type and a 1485-bp for the odc-1(pc13::Tc1) allele. PCR using the oligonucleotides SMD-1-S and SMD-1-AS leads to fragments for wild-type and the smd-1(tm2772) allele of 942 bp and 164 bp, respectively. K07-S2 and K07-AS2 were used to distinguish the wild-type (971 bp) and catp-5(mun1) allele (472 bp).

Identification of toxic polyamines

C. elegans N2 wild-type L1 larvae were transferred to NGM plates supplemented with various polyamines and polyamine analogs at a final concentration of 5 mM. Growth was followed for 5 d.

Norspermidine toxicity assays

To examine a stage-specific effect of norspermidine on C. elegans, N2 worms of a synchronous population were exposed to 5 mM norspermidine at different stages of their life cycle. Their development was monitored, and the number of progeny was determined.

Norspermidine tolerance assays were performed as follows: known numbers of C. elegans eggs or, in case of rescue strains, L2 larvae exhibiting the rol-6 phenotype were picked on NGM plates supplemented with increasing concentrations of norspermidine (0 to 5 mM). To analyze the effect of natural polyamines on norspermidine toxicity, NGM plates additionally contained an equimolar concentration of spermidine or putrescine. The development of the worms was monitored, and on d 4, the percentage of animals that had become adults was determined.

RNAi efficiency assay

RNAi assays were carried out as described by Kamath and Ahringer (9) with minor modifications. Synchronous C. elegans L4 larvae were transferred to NGM plates containing 2.5 mM IPTG and 50 μg/ml ampicillin that had been previously inoculated with an E. coli HT115 RNAi marker strain, and worms were incubated for 72 h at 15°C. The HT115 marker strains produce dsRNA corresponding to the C. elegans genes dpy-6 and bli-4 (Geneservice, Cambridge, UK). F1 eggs were transferred to RNAi plates supplemented with increasing concentrations of norspermidine (0 to 3 mM) or with 3 mM norspermidine and an equimolar concentration of spermidine. Animals were cultured for an additional 3 d at 20°C. The percentage of adult F1 worms that showed the expected phenotype was determined.

Chemistry-to-gene screen

The norspermidine-resistant C. elegans mutant strain nor-2 was isolated in a Bristol N2 background by applying standard ethyl methanesulfonate mutagenesis methodology (10, 18). Mutagenized worms were grown under standard conditions, before F2 eggs were spread on 25 NGM plates containing 5 mM norspermidine. Following an incubation of 5 d, healthy living animals (only 1 worm/plate) were individually transferred to new 5 mM norspermidine selection plates. Prior to further analyses, the isolated resistant strain nor-2 was outcrossed 10 times to N2, whereby F2 eggs of each cross were reselected on selection plates. Mapping was performed by crossing nor-2 males into DA438, CB4856, and EM122, respectively, selecting the F2 animals on 5 mM norspermidine. Snip-single nucleotide polymorphism (SNP) mapping was carried out as described by Wicks et al. (20). The catp-5(mun1) allele of nor-2 was identified by PCR using the gene-specific oligonucleotides K07-S2 and K07-AS2.

Plasmid constructs

catp-5(3.9)::gfp

A 3.9-kb promoter fragment of catp-5 including the first exon of 6 bp was amplified from the C. elegans cosmid K07E3 using the Expand Long Template PCR system (Roche, Mannheim, Germany) and the primers K07-Res-S and K07-GFP-AS5. The PCR product was cloned into pPD95.77 provided by A. Fire (Carnegie Institute, Baltimore, MD, USA).

catp-5(6.2)::gfp

A 6.2-kb promoter fragment of catp-5 including the putative alternative second translational start site was amplified by PCR using the primers K07-Res-S and K07-GFP-AS5.2 and cloned into pPD95.77.

catp-5(12.2)::gfp

A 12.2-kb promoter fragment including the entire ORF of catp-5 was amplified by PCR using the primer pair K07-Res-S and K07-GFP-AS-Sma. The PCR product was cloned into pPD95.77.

catp-5(overex)

A 13.2-kb genomic fragment corresponding to 3892 bp of the 5′ upstream region, the entire set of introns and exons, and 989 bp of the 3′ region of the catp-5 gene was amplified by PCR from the C. elegans cosmid K07E3 using the primers K07-Res-S and K07-Res-AS. Template DNA was digested for 1 h with 10 U DpnI, and the PCR product was column purified.

Transgenic C. elegans

Germline transformation was performed by coinjecting vector constructs (20–50 μg/ml) with the pRF4 plasmid (80 μg/ml) encoding the dominant marker gene rol-6 into the germline of young adults (21). N2 wild-type worms were injected with catp-5(3.9)::gfp and catp-5(6.2)::gfp. The nor-2 mutant carrying the catp-5(mun1) allele was transformed with catp-5(12.2)::gfp and catp-5(overex) for rescue experiments. To investigate the cell-specific, developmentally regulated transcription of catp-5, GFP expression patterns were analyzed by fluorescence microscopy.

Analyses of CATP-5 expression by RT-PCR

Total RNA was extracted from C. elegans N2 wild-type and catp-5(mun1) mutant worms applying TRIzol reagent (Invitrogen, Karlsruhe, Germany). Subsequently, RNA was used in reverse transcriptions (Fermentas, St. Leon-Rot, Germany). cDNA products served as templates in PCR reactions using the primer pairs ACT-1-S and ACT-1-AS for C. elegans actin-1 and K07E3.7-S(c880) and K07E3.7-AS(c1864) for C. elegans catp-5. The oligonucleotides for catp-5 anneal on either side of the deletion allele catp-5(mun1) found in nor-2, resulting in theoretical PCR products of 985 bp for wild type and 604 bp for nor-2, respectively.

Polyamine determination

Synchronized 3-d-old young adult worms were sonicated, and the 10,000 g supernatant was deproteinized by adding 0.2 N perchloric acid. Probes were derivatized with dansyl chloride (2 mg/ml acetone), before being separated by high-pressure liquid chromatography (Thermo Scientific, Dreieich, Germany) on a nucleosil 120–5 C18 column (250 × 3 mm; Macherey and Nagel, Düren, Germany), according to Kabra et al. (22). Dansylated polyamines were detected by fluorescence spectrophotometry (Jasco 821-FP; Thermo Scientific).

Norspermidine accumulation assay

Synchronized young adult C. elegans were transferred to 5 ml NGM liquid medium containing 5 mM norspermidine and penicillin (50 U/ml)/streptomycin (50 μg/ml). Following overnight incubation at 20°C, worms were washed 4 times in 50 ml ice-cold M9 buffer and prepared for polyamine determination.

Uptake of fluorescent polyamines conjugates

Mixed staged worms were moved to 250 μl M9 buffer supplemented with 2 mM of the fluorescent polyamine conjugate N1-(anthracen-9-ylmethyl)homospermidine (Ant-4,4) (Fig. 1A) (23). After incubating for 1 h at 20°C, worms were washed twice in ice-cold M9 buffer containing 50 mM spermidine and twice in M9 only. Uptake of the polyamine conjugates was examined by fluorescence microscopy. Controls were kept in the presence of 10 mM spermidine at 4°C or in M9 buffer only.

Figure 1.

Figure 1.

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Biological effects of norspermidine. A) Chemical structures of norspermidine, spermidine, spermine, and Ant-4,4. B) Screening for toxic polyamines. Ten eggs of C. elegans N2 wild type were transferred to NGM plates supplemented with 5 mM polyamines, as indicated. Growth was monitored for 6–9 d (control plates without additional polyamines). N2 wild-type worms cultured for 4 d on control plates developed to adult hermaphrodites, whereas in the presence of 5 mM norspermidine, development was blocked at L2/L3 stage (single-worm pictures). NSPD, norspermidine; PUT, putrescine; SPD, spermidine. Scale bar = 100 μm). C) Norspermidine toxicity assay. Eggs of C. elegans wild-type (▪) and nor-2 worms carrying the catp-5(mun1) allele (▾) were moved to NGM plates supplemented with increasing concentrations of norspermidine. Their development was monitored for 4 d. In the case of the rescue lines catp-5(mun1);catp-5(+) (▿) and catp-5(mun1);catp-5(+)::gfp (⋄), worms were placed on selection plates at L2, allowing the identification of the rol-6 marker phenotype. Percentages of animals that became adult are shown (means of 4 independent experiments; each data point represents ≥80 worms). Inset: percentage of N2 wild-type worms that developed to adult animals on 5 mM norspermidine NGM-plates supplemented with and without equimolar concentrations of spermidine and putrescine, respectively. catp-5(mun1) worms on 5 mM norspermidine plates served as a control. (n=50 animals). D) RNAi efficiency assay. Adult C. elegans N2 wild-type and nor-2 mutant worms carrying the catp-5(mun1) allele were transferred from initial RNAi feeding plates onto RNAi feeding plates supplemented with increasing concentrations of norspermidine. Percentages of F1 animals that exhibited the expected phenotype were scored. Right bar in each graph depicts the results when plates contained 3 mM norspermidine along with 3 mM spermidine. RNAi clones targeting dpy-6 (left panel) and bli-5 (right panel) were used as indicators of RNAi efficiency. Data are means ± se of ≥4 independent experiments. Bars represent ≥80 animals. **P <0.01, ***P<0.001 vs. wild type; Student’s t test).

Determination of developmental parameters

For the determination of postembryonic development, gravid adult worms were put on fresh plates and allowed to lay eggs for 3–4 h. Adults were removed, and F1 animals were transferred to individual plates, once they reached L3 stage. The time point of the first egg deposition was monitored. To determine brood sizes, worms were cultured individually on NGM plates. Adults were transferred daily to new plates until egg production ceased. Plates with progeny were retained and counted when larvae reached L3/L4. Life-span assays were carried out at 20°C. In each experiment, 50 eggs were equally distributed onto 5 plates. Worms were examined daily and were scored as dead when they no longer responded to touch-provoked movement. Animals that crawled away from plates were replaced by worms from a parallel substitute plate. Body size of worms was measured by using the free image-processing program ImageJ (National Institutes of Health, Bethesda, MD, USA) as described by Mörck and Pilon (24). Synchronized worms were incubated at 20°C, and body length and width were examined daily.

RESULTS

Norspermidine impairs growth of C. elegans but not of E. coli

To find a selection compound for our chemistry-to-gene screen strategy (see below), we tested the effect of polyamines and polyamine analogues on C. elegans cultures. Spermine and the structural spermidine analog norspermidine (Fig. 1A) were found to be toxic to C. elegans, affecting its growth at a concentration of 5 mM (Fig. 1B). Because standard continuous culturing of C. elegans depends on E. coli OP50 as a food source, we next examined the effect of these polyamines on bacterial growth. While spermine also blocked the proliferation of E. coli OP50, the growth rate of the food bacteria was not significantly altered in the presence of norspermidine (Supplemental Fig. 1), implicating that the spermidine analog was a suitable compound for our approach.

Further analyses revealed that C. elegans wild-type eggs, when exposed to 5 mM norspermidine, did not develop to adult worms, but mostly remained at the L2/L3 stage without forming dauer diapause, the alternative third larval stage of C. elegans that is usually induced by unfavorable conditions (Fig. 1B). If worms were moved to norspermidine-containing plates at any later stage of their development, they grew normally to adults. However, depending on the developmental stage, the fecundity of these worms was distinctly affected. The earlier the worms were transferred, the smaller the brood size (Supplemental Table 2). As shown by drug sensitivity assays, C. elegans was affected by norspermidine in a dose-dependent manner (Fig. 1C). Egg-to-adult development was completely blocked at 3 mM of the toxic polyamine. Exposure to similar concentrations of the physiological polyamine spermidine had no obvious negative effects on C. elegans. In fact, equimolar spermidine notably mitigated growth inhibition caused by norspermidine (Fig. 1B, C). Under these conditions, worms grew continuously; however, their postembryonic development and their growth rate were slightly affected. Remarkably, the growth inhibitory effect of norspermidine on C. elegans was hardly affected by putrescine.

Norspermidine affects RNAi efficiency in C. elegans

Owing to their polycationic character, a great portion of the intracellular polyamines were previously found to be bound to nucleic acids (25), being involved in the regulation of several biochemical processes where nucleic acids participate (2, 26). Because cytotoxic polyamine analogues are suggested to function by disturbing the intracellular polyamine homeostasis (1, 3), we investigated the effect of norspermidine on the RNAi process in C. elegans. For this reason, we developed an RNAi efficiency assay. RNAi efficiency was analyzed by employing two RNAi constructs, corresponding to the genes dpy-6 and bli-5 as markers. When providing dsRNA by the feeding method under standard conditions, ≥90% of RNAi-treated worms were scored positive for exhibiting the respective phenotype (Fig. 1D). However, the addition of increasing concentrations of norspermidine to the culture medium drastically reduced the numbers of worms that were affected by RNAi treatment in a dose-dependent manner. At 3 mM norspermidine, <10% of the RNAi worms had the expected phenotype. Again, supplementing the medium with equal concentrations of spermidine abrogated the inhibitory effect (Fig. 1D). Remarkably, a similar inhibitory effect on the RNAi process by norspermidine was obtained, when dsRNA was introduced by microinjection (Supplemental Fig. 2A).

Isolation of the norspermidine-resistant C. elegans strain nor-2

A forward genetic screen was performed for mutants that are able to grow in the presence of toxic concentrations of norspermidine. We hypothesized that this chemistry-to-gene screen strategy should provide us with C. elegans mutants whose tolerance toward the toxic spermidine analog is achieved by an impaired polyamine transport system, although other resistance mechanisms are also possible. Following standard ethyl methanesulfonate mutagenesis in a N2 genetic background, the strain nor-2 was isolated. The norspermidine-resistant phenotype of nor-2 is manifested by at least two features. First, in contrast to N2 wild-type worms, the egg-to-adult development of nor-2 was not significantly affected by norspermidine concentrations of up to 5 mM (Fig. 1C), and the strain could be continuously cultured under these conditions. Second, the negative effect of increasing concentrations of norspermidine on RNAi efficiency observed for N2 wild-type was markedly reduced in nor-2 worms (Fig. 1D). Moreover, nor-2 was found to be cross resistant to spermine (data not shown). Further examination revealed that nor-2 mutants appeared healthy and did not display any obvious abnormalities.

A deletion in catp-5 confers norspermidine resistance

To identify the mutated gene locus that is responsible for the increased norspermidine tolerance of the nor-2 strain, standard mapping and linkage analyses were applied. Norspermidine resistance was linked to chromosome X and was found to be located between the two visual markers dpy-6 (X:-0.00 cM) and unc-18 (X:-1.37 cM). Its position was further refined by SNP mapping to the region between the SNP markers F45E1[1] (X:-0.77 cM) and K07E3[4] (X:-0.53 cM). According to the C. elegans genome project (http://www.wormbase.org), this region consists of 21 genes, including the K07E3.7 gene of a putative cation P-type ATPase, termed catp-5. PCR and subsequent DNA sequencing analyses using genomic DNA from wild type and nor-2 revealed a 499-bp deletion in the catp-5 gene of the mutant strain (Fig. 2A). The role of the identified catp-5(mun1) allele in norspermidine resistance was confirmed by rescue experiments. Overexpression of wild-type copies of catp-5 in the catp-5(mun1) background not only abrogated norspermidine resistance but led to elevated norspermidine sensitivity (Fig. 1C).

Figure 2.

Figure 2.

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Genetic organization and expression of C. elegans catp-5. A) C. elegans N2 wild-type and norspermidine-resistant nor-2 mutant worms were analyzed for their genotype by single-worm PCR analyses. A part of the gene catp-5 was amplified by using oligonucleotides flanking the deletion present in the catp-5(mun1) allele of nor-2 (see Materials and Methods). B) RT-PCR analyses of C. elegans N2 wild-type and catp-5(mun1) animals. Oligonucleotides flanking the deletion of catp-5 were used in the PCR reaction (see Materials and Methods). No catp-5 PCR product was obtained for the catp-5(mun1) allele. Owing to the deletion identified at the genomic level, the theoretical size of the catp-5 PCR fragment in catp-5(mun1) worms is 612 bp. Band of ∼900 bp corresponds to the internal control PCR fragment of the act-1 gene. C) Genomic organization of C. elegans catp-5 and reporter gene constructs used in microinjection. Position of the deletion allele catp-5(mun1) identified in the nor-2 strain is shown. D) Expression pattern of C. elegans CATP-5. Transgenic animals carrying the construct catp-5(3.9)::gfp (a) or catp-5(6.2)::gfp (b) exhibited cytosolic GFP signals with the same spatial and temporal expression pattern. In transgenic animals carrying catp-5(12.2)::gfp (c–h), which includes the complete ORF of catp-5, fluorescent signals are associated with the excretory cell (e, f) and the apical membrane of the intestinal cells (g, h). Scale bars = 100 μm (a–d); 50 μm (e–h).

CATP-5 is a P5B-type ATPase

C. elegans CATP-5 codes for a member of the P-type ATPase superfamily, which encompasses a large number of membrane proteins involved in active transport processes of charged substrates like metal ions, protons, or phospholipids across biological membranes (27). In silico analyses revealed that the topology of C. elegans CATP-5 resembles the architecture of the known P-type ATPases to a great extent. For example, the conserved cytosolic P-type ATPase amino acid motif DKTGT (Fig. 3A), the Asp residue of which is phosphorylated during catalysis (27), is present. C. elegans CATP-5 was predicted to have 11 transmembrane helices, leading to a topology with an extracellular N- and intracellular C-terminus that is untypical for P5-type ATPases (Fig. 3B).

Figure 3.

Figure 3.

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C. elegans CATP-5, a P5B-type ATPase that operates in polyamine transport. A) Amino acid sequence comparison of P5-type ATPases. The highly conserved part of the P domain that carries the P-type ATPase-specific Asp residue (shaded in gray, in C. elegans CATP-5 Asp403) is aligned. Asterisks indicate amino acid residues that are conserved in ≥9 sequences. Selected sequences encompass the 4 annotated P5-type ATPases of C. elegans CATP-5 (K07E3.7), CATP-6 (W08D2.5), CATP-7 (Y59H11AR.2a), and C10C6.6, as well as Saccharomyces cerevisiae Cod31p/Spf1p (ScSpf1p), Arabidopsis thaliana MIA (AtMIA), and the human P5-type-ATPases HsATP13A3 and HsATP13A4. For comparison, the SERCA1 of Rattus norvegicus, a P2A-type-ATPase, is listed in the bottom line. Amino acid sequences were aligned by ClustalW 1.83 (http://www.ebi.ac.uk/Tools/clustlw). B) Putative schematic topology of C. elegans CATP-5. Topology prediction was carried using TMHMM 2.0 (http://www.cbs.dtu.dk/services/TMHMM-2.0/). C) Uptake of the fluorescent polyamine conjugate Ant-4,4 by C. elegans wild type. N2 wild-type worms take up Ant-4,4 efficiently, as indicated by the substantial fluorescence appearing within the fluid-filled pseudocoelomic cavity (solid arrowheads), which contains the intestine and other internal organs. In contrast, the relatively strong autofluorescence of the intestinal cells (arrows) largely prevents the visualization of Ant-4,4 in this organ. This implies that Ant-4–4, once it has been taken up by the intestine, is transferred into the pseudocoelomic fluid, which carries out blood-like functions such as intercellular nutrient transport. Uptake is drastically diminished at 4°C or by coadministration of 10 mM spermidine, since Ant-4,4 signals within the worm are no stronger or only slightly stronger than the mainly intestinal autofluorescence of control worms kept in M9 (shown in D). D) Uptake of Ant-4,4 is CATP-5 dependent. In catp-5(mun1) mutants, fluorescence is restricted mostly to the gut lumen (open arrowheads), and only weak fluorescence is seen within the pseudocoelom (solid arrowheads). catp-5(mun1) animals rescued by catp-5(12.2)::gfp are stained as wild-type worms. Arrows indicate intestinal cells. Anterior parts of the animal are shown. Scale bars = 100 μm.

P-type ATPases are divided into five related subclasses, designated P1 to P5. A Basic Local Alignment Search Tool (BLAST) search using the C. elegans CATP-5 sequence as query revealed several P5-type ATPases (alignment, see Supplemental Fig. 3), whose members have been recently subdivided into subtypes P5A and P5B (28). The highest similarity was found with the potential P5B-type ATPases C. elegans CATP-6 and CATP-7 (score 40), followed by the human ATPase13A3 (score 35) (29) and ATP13A2 (score 33) (30). The similarity to known P5A-type ATPases such as MIA from Arabidopsis thaliana (score 20) (31) or Cod31p/Spf1p from yeast (score 21) (32) was less significant.

The C. elegans catp-5 gene spans ∼8.3 kb from the first ATG initiator site to the termination codon. According to the cDNA clones yk1632 and yk1623, obtained from the C. elegans EST project (courtesy of Yuji Kohara, National Institute of Genetics, Mishima, Japan), it produces two distinct transcripts, presumably by alternative splicing. The open reading frames (ORFs) of these two mRNAs differ in the alternative use of N-terminal exons. The longer transcript, with an ORF of 3612 bp, carries a spliced leader 1 and is encoded by 17 exons. The smaller transcript has an ORF of 3525 bp and is encoded by 15 exons. Consequently, two isoforms, CATP-5a and CATP-5b, are expected to occur in C. elegans, consisting of 1174 and 1203 amino acid residues, respectively (Supplemental Fig. 4). Isoform CATP-5a has a deduced molecular mass of 131.3 kDa, CATP-5b of 134.7 kDa.

Figure 4.

Figure 4.

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C. elegans catp-5 interacts with odc-1(pc13) and smd-1(tm2772). Adult body volume (A, B) and adult life span (C) of N2 wild-type (▪), catp-5(mun1) (▿), odc-1(pc13) (▴), smd-1(tm2772) (▵), catp-5(mun1);odc-1(pc13) (•), and catp-5(mun1);smd-1(tm2772) animals (○).

The catp-5(mun1) allele contains a deletion of 499 bp, which results in the loss of exon IX (with respect to CATP-5b) (Fig. 2C) that encodes the predicted transmembrane helices M4 and M5, as well as the interjacent extracellular region (Fig. 3B). Furthermore, the subsequent ORF is destroyed. Hence, the sequence lacks crucial domains putatively involved in the transport process (27) and very likely represents a loss-of-function or null mutant. To verify this, expression of CATP-5 mRNA was examined by RT-PCR (Fig. 2B). PCR products of the expected size corresponding to CATP-5 were obtained only for C. elegans wild-type and not for catp-5(mun1) animals. Control actin transcript was detected in both worm lines.

CATP-5 expression pattern

The spatial, temporal, and subcellular expression pattern of CATP-5 was analyzed by a reporter gene approach in which expression of the green fluorescent protein (GFP) was placed under the control of different catp-5 promoter sequences. Since CATP-5 is a putative transmembrane protein with two predicted isoforms, we used three constructs that were introduced into C. elegans by microinjection: catp-5(3.9)::gfp, which contains only the first two codons of the larger isoform CATP-5b; catp-5(6.2)::gfp, which is extended to the second translational start site of the isoform CATP-5a; and catp-5(12.2)::gfp, which spans the entire ORF of both isoforms (Fig. 2C). For all transgenic worm lines, fluorescence was detected in the 20 intestinal cells and in the excretory cell (Fig. 2Da–h). GFP expression was present at all larval stages and in adult worms, but was not found in embryos. Although no alteration in spatial and temporal expression was observed, the catp-5(6.2)::gfp construct led to a brighter fluorescence in all cells, when compared to worms that carried the catp-5(3.9)::gfp construct (Fig. 2Da, b). Inclusion of the entire catp-5 ORF and hence the complete set of predicted transmembrane helices in the catp-5(12.2)::gfp construct led to a membrane-bound GFP signal (Fig. 2Dc). Remarkably, the intestinal fluorescence was associated exclusively with the apical membrane that faces the gut lumen (Fig. 2Dg, h).

CATP-5 promotes uptake of fluorescent polyamine conjugates

To monitor polyamine uptake by C. elegans, we used the fluorescent polyamine conjugate Ant-4,4, which has been previously shown to be accepted by the mammalian polyamine uptake system (23). N2 wild-type worms took up the polyamine conjugate efficiently at 20°C, as indicated by the fluorescent signal within the pseudocoelom (Fig. 3C), Ingestion of Ant-4,4 was not observed at 4°C and was drastically reduced in the presence of 10 mM spermidine (Fig. 3C). When catp-5(mun1) worms were exposed to Ant-4,4, fluorescent signals were almost completely restricted to the gut lumen (Fig. 3D), indicative of drastically diminished absorption by worms lacking CATP-5. Again, overexpression of CATP-5 in the catp-5(mun1) background restores the wild-type situation (Fig. 3D).

catp-5(mun1) animals accumulate less toxic polyamines

Since CATP-5 has been found to operate in polyamine uptake, we next examined whether the intracellular polyamine pool was altered in catp-5(mun1) worms. The polyamine pattern of catp-5-deficient animals cultured under standard conditions was similar to that of N2 wild-type worms (Table 1). Spermidine represented the major polyamine, and the spermidine to putrescine ratio was estimated to be ∼2:1. To analyze the physiological basis for the increased tolerance toward toxic polyamines, catp-5(mun1) and N2 wild-type worms were incubated for 24 h in the presence of 5 mM norspermidine before their polyamine content was determined. Again, the levels of the natural polyamines were similar in both strains. However, catp-5(mun1) worms accumulated significantly less of the toxic polyamine than N2 wild-type worms (Table 1). Hence, norspermidine exceeded the intracellular spermidine level in N2 wild-type with a ratio of 1.6:1, whereas the norspermidine to spermidine ratio in animals carrying the catp-5(mun1) allele was calculated to be 1:1.6.

TABLE 1.

Polyamine concentrations in C. elegans

Strain Putrescine Norspermidine Spermidine n
Synchronous young adult C. eleganscultured under standard conditions
Wild type 97.7 ± 5.0 — 178.1 ± 9.9 6
catp-5(mun1) 98.0 ± 5.6 — 178.2 ± 6.7 7
odc-1(pc13) 68.8 ± 3.3 — 113.9 ± 6.2 7
smd-1(tm2772) 129.1 ± 11.2 — 115.6 ± 11.9 6
catp-5(mun1);odc-1(pc13) 23.1 ± 3.1 — 65.4 ± 5.2 5
catp-5(mun1);smd-1(tm2772) 218.5 ± 8.7 — 18.6 ± 2.2 3
Incubation for 24 h in NGM liquid medium supplemented with 5 mM norspermidine
Wild type 70.5 ± 10.6 96.1 ± 7.2 59.3 ± 7.0 4
catp-5(mun1) 76.8 ± 6.7 38.9 ± 9.0 63.1 ± 11.9 4
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Values represent means ± se (nmol/mg protein). 

Genetic interaction of catp-5 with the polyamine synthesis genes odc-1 and smd-1

To further investigate the role of CATP-5 in polyamine metabolism, we performed genetic interaction studies with catp-5(mun1) using two C. elegans mutant strains with defects in the key enzymes of polyamine synthesis, odc-1(pc13) (12, 13) and smd-1(tm2772). smd-1(tm2772) is the first available and up to now uncharacterized C. elegans AdoMetDC allele. PCR and sequence analyses confirmed the reported 778-bp deletion (http://www. wormbase.org) leading to a smd-1 loss-of-function or null mutant (for details see Supplemental Fig. 5). Like the odc-1(pc13) loss-of-function mutant, smd-1 deficient worms were found to be viable under standard culture conditions. Moreover, they resemble the phenotype of odc-1-deficient worms, by producing a smaller number of progeny (Table 2) and by having a body size slightly smaller than that of wild-type worms (Fig. 4A, B). The sole function of AdoMetDC is the production of decarboxylated S-adenosylmethionine for spermidine synthesis, acting in parallel to putrescine-providing pathways. Consistent with the loss of AdoMetDC function, the spermidine level of smd-1(tm2772) was found to be significantly reduced by 35%, when compared to wild-type worms (P<0.0001, t test) (Table 1), and putrescine derived from ODC activity and/or uptake could not be further used in spermidine synthesis, but accumulated, leading to 1.3-fold elevated putrescine concentrations (P<0.001). In the C. elegans odc-1(pc13) loss-of-function mutant, putrescine and spermidine concentrations were reduced by ∼30 and 35%, respectively (P<0.0001) (Table 1).

TABLE 2.

Postembryonic development and number of progeny

Strain PE (h) N2 (%) Number of progeny N2 (%)
N2 wild type 68.1 ± 0.3 (105) − 259 ± 4 (141) −
catp-5(mun1) 68.3 ± 0.5 (48) 100 212 ± 4 (119) 82
odc-1(pc13) 70.8 ± 0.3 (124) 104 127 ± 5 (78) 49
smd-1(tm2772) 66.0 ± 0.2 (88) 97 153 ± 5 (102) 59
catp-5(mun1);odc-1(pc13) 114.4 ± 1.8 (46) 168 70 ± 5 (42) 27
catp-5(mun1);smd-1(tm2772) 92.0 ± 1.0 (145) 135 62 ± 4 (41) 24
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Values are means ± se with number of animals in parentheses. PE, postembryonic development. 

It has been shown in previous studies that the C. elegans ODC loss-of-function mutant proliferates only when exogenous polyamines are available (13). As CATP-5 is involved in polyamine uptake, one would expect that worms carrying homozygous mutations for one of the polyamine synthesis enzymes in the catp-5(mun1) genetic background should exhibit even more drastic effects on the polyamine pattern than the respective single mutants. Accordingly, catp-5(mun1);odc-1(pc13) worms were found to have a markedly reduced putrescine level, representing only 24% of the putrescine value of the wild-type (P<0.0001) (Table 1). Spermidine concentration was also affected and decreased by ∼60% (P<0.0001). Most drastic was the alteration in catp-5(mun1);smd-1(tm2772) worms (Table 1), in which the spermidine level dropped to 10% (P<0.0001) when compared with wild-type worms. However, the putrescine concentration was found to be extremely enhanced by >2-fold (P<0.005), which is consistent with the inability of these worms to convert putrescine into spermidine.

The double mutants catp-5(mun1);odc-1(pc13) and catp-5(mun1);smd-1(tm2772) are characterized by significantly reduced brood sizes (Table 2). The reduced number of progeny is most likely attributable to decreased egg production, since L1 larvae hatched from >90% of the laid eggs (data not shown). Both double mutants exhibit delayed postembryonic development (Table 2), which was not found for the respective single mutants. At 20°C, they became adults 4–5 d after eggs had been laid. As outlined above, odc-1(pc13) and smd-1(tm2772) displayed slightly smaller body sizes, when compared with wild-type and catp-5(mun1) worms (Fig. 4A, B). Again, the effect was more severe in the double mutants catp-5(mun1);odc-1(pc13) and catp-5(mun1);smd-1(tm2772). Furthermore, the mean adult life span of both double mutants was drastically reduced by almost 50%, while odc-1(pc13), smd-1(tm2772), and catp-5(mun1) single mutants had a mean adult life span similar to that of N2 wild-type (Fig. 4C and Table 3).

TABLE 3.

Adult life span

Strain Adult life span (d)
N P valuea
Mean Max
N2 wild type 14.8 ± 0.4 24 285 −
catp-5(mun1) 14.2 ± 0.3 24 150 0.04
odc-1(pc13) 14.9 ± 0.9 29 175 0.90
smd-1(tm2772) 14.4 ± 0.4 27 185 0.20
catp-5(mun1);odc-1(pc13) 8.2 ± 0.7 16 130 <0.001
catp-5(mun1);smd-1(tm2772) 8.6 ± 0.3 17 175 <0.001
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Values are means ± se (n≥3). N = total number of animals. 

a

Comparison of mean life span of mutants and N2; Student’s ttest. 

DISCUSSION

The maintenance of adequate intracellular polyamine levels is of central importance to organisms, given the often lethal consequences of a disturbed polyamine metabolism. Apart from biosynthesis, most organisms are able to replenish their polyamine pool by uptake from the diet. However, the molecules that account for metazoan (including mammalian) polyamine transport are still largely unknown (2, 3, 5). By employing the nematode C. elegans as a multicellular genetic model in a chemistry-to-gene screen, we have identified the novel P5B-type ATPase CATP-5. Our data suggested that CATP-5 either is a polyamine transporter or operates as a positive regulator of polyamine uptake in C. elegans.

The biological effects of polyamine analogues can be often explained by an antagonistic mode of action, replacing the natural polyamines, but without fulfilling their function (1,2,3). Accordingly, our data on the resistant catp-5(mun1) mutant allele strongly suggest that the toxic effect of norspermidine on C. elegans is most probably related to high intracellular norspermidine to spermidine ratios. During C. elegans life cycle, oogenesis/embryogenesis and larval development were found to be sensitive toward norspermidine treatment. Interestingly, egg production and embryonic development of C. elegans have been previously demonstrated to require polyamines (13). Norspermidine was also reported to be toxic for mammalian cells (33). Hence, it is quite astonishing that it had no negative effect on E. coli growth, but, on the contrary, was previously shown to even promote growth of E. coli mutants that lack polyamine synthesis (34).

Another remarkable feature of norspermidine elucidated in the present study is its inhibitory effect on RNAi in C. elegans. In principle, possible targets of norspermidine are the production of dsRNA in E. coli, the uptake of dsRNA by C. elegans, the systemic spreading of dsRNA, and the processing of dsRNA within the nematode. The fact that catp-5(mun1) worms were found to be RNAi susceptible, even in the presence of otherwise inhibitory concentrations of the spermidine analog, excludes impaired dsRNA production in E. coli. Furthermore, the RNAi response was similarly reduced by norspermidine, irrespective of whether dsRNA was provided by feeding, which requires the ingestion of dsRNA via the intestine (35), or by microinjection, which is independent of intestinal transport. This result indicates that norspermidine does most probably not target the uptake of dsRNA by C. elegans. In addition, norspermidine did not affect systemic RNAi (also known as non-cell-autonomous RNAi) in the nematode (see Supplemental Fig. 2B). Therefore, our data give strong evidence for the conclusion that norspermidine targets the RNAi process within C. elegans. Accordingly, catp-5(mun1) worms that accumulated less of exogenously administered norspermidine than wild-type worms exhibited a higher tolerance in their RNAi response toward the spermidine analog. The nonphysiological analog norspermidine may disturb the RNAi machinery, probably by displacing the natural polyamines from binding sites and in this way affecting RNA conformation and/or RNA-RNA and RNA-protein interactions that characterize the course of the RNAi process (35). In fact, the double-mutant catp-5(mun1);smd-1(tm2772) that contains a reduced spermidine level exhibited an enhanced sensitivity toward norspermidine in its RNAi response (Supplemental Fig. 2C). Although the reduced spermidine level of catp-5(mun1)-smd-1(tm2772) worms did not affect RNAi efficiency in the absence of the analog, the observed antagonistic activity of norspermidine points to an involvement of the natural polyamines in RNAi. In this regard, it is interesting that intracellular spermidine has been only recently reported to bind to dsRNA with high affinity. It was suggested that spermidine predominantly interacts with RNA rather than with DNA and that binding to RNA, in particular to bulged-out regions of dsRNA, regulates several biological processes by stabilizing and/or changing RNA structures (36).

C. elegans catp-5 encodes a P5B-type ATPase. Apparently, P5-type ATPases are restricted to eukaryotes, where they are widely distributed (28). Best investigated are the P5-type ATPases A. thaliana MIA and yeast Spf1p/Cod1p, which were reported to be functionally conserved (31, 32), although their substrate specificity has not yet been revealed. Our own results (data not shown) and phylogenetic analyses by Møller et al. (28) indicated that C. elegans CATP-5 does not branch with these transporters but is more closely related to the C. elegans proteins CATP-6 and CATP-7, as well as to the mammalian P5-type ATPases ATP13A2, ATP13A3, and ATP13A4. Accordingly, the P5-type ATPases have been divided into the subfamilies P5A and P5B (28). Human ATP13A2 has been reported to be associated with the Kufor-Rakeb syndrome, a hereditary Parkinson’s disease (30). Remarkably, C. elegans P5-type ATPase CATP-6, the closest homologue of CATP-5, has been recently identified in an RNAi screen for genes involved in stabilization of a synthetic α-synuclein, the misfolding of which is associated with human Parkinson’s disease, therefore representing a putative functional homologue of ATP13A2 (37). Human ATP13A3 was cloned from senescent parenchymal kidney cells and was discussed to be involved in cellular aging (29). These reports indicate that P5B-type ATPases are linked to important biological processes and to human diseases. However, functional analyses have not been performed on these human transporters; hence, their precise physiological roles are still unresolved.

C. elegans CATP-5 is expressed in the apical membrane of the intestine and in the excretory cell, making it the first P5-type ATPase, to our knowledge, that is associated with the plasma membrane. This spatial and subcellular expression pattern is consistent with the proposed transport function. Both organs are responsible for the regulation of ion and metabolite homeostasis at the organismic level harboring numerous transporters and high transport activities. Several lines of evidence suggest that CATP-5 is a polyamine transporter. In particular, the redundant function of CATP-5 and polyamine synthesis in polyamine homeostasis and the fact that uptake of the polyamine conjugate Ant-4,4, an established model metabolite to study mammalian polyamine transport (23), depends on the presence of CATP-5 strongly support this notion. However, worms carrying the loss-of function allele catp-5(mun1) and the double-mutant catp-5(mun1);smd-1(tm2772) still accumulate low levels of norspermidine and spermidine, respectively. Since the mechanism of action has not been established for any P5-type ATPase, further studies will be necessary to definitively elucidate whether CATP-5 has polyamine transport function by itself and a parallel transport system for polyamines is present in C. elegans, or whether CATP-5 facilitates the function of an associated polyamine transporter.

Polyamine transporter genes have been identified in prokaryotic and eukaryotic unicellular organisms (6,7,8). However, all information on these molecules did not help to elucidate the polyamine transporter genes in metazoa, suggesting a distinct molecular mechanism. It is still a matter of debate whether polyamines are taken up by an endocytosis process (38) or by a classical transporter (39). Recently, polyamine transporters have been biochemically and pharmacologically characterized in S2 and epithelium cells from Drosophila melanogaster, respectively (17, 40). They were found to share many properties with mammalian polyamine transport, implying that the molecular mechanism of polyamine uptake may be conserved among metazoan organisms.

The eukaryotic polyamine transport system is of pharmacological importance, since polyamine analogues and polyamine-drug conjugates enter cells via this route (1,2,3,4,5). Polyamine analogues target the natural polyamines and their metabolism, thereby affecting cell proliferation and growth. Polyamine-conjugates facilitate the uptake of otherwise poorly transportable drugs attributable to their polyamine backbone. However, owing to the lack of molecular information of the polyamine transport system, up to now, optimization of chemotherapeutic polyamine structures was limited to biochemical and pharmacological approaches. The identification of C. elegans CATP-5 as a potential polyamine transporter may now offer the opportunity to identify polyamine transport systems in other metazoan organisms making the rational design of selective polyamine compounds feasible. Furthermore, the C. elegans mutant allele catp-5(mun1) is suggested to represent a powerful model to test polyamine-based chemotherapeutics at a metazoan level.

The unchanged polyamine pattern of catp-5(mun1) mutants indicates that CATP-5 is dispensable for polyamine homeostasis in worms that contain a functional polyamine biosynthesis. Similarly, mutant worms that lack one of the polyamine synthesis key enzymes odc-1 or smd-1 contain only moderately altered polyamine levels, alluding to other pathways, most likely uptake from diet, that compensate for the loss of synthesis activity. Accordingly, analyses of the double mutants catp-5(mun1);odc-1(pc13) and catp-5(mun1);smd-1(tm2772) demonstrated that polyamine synthesis enzymes interact synergistically with CATP-5. Previous studies have clearly demonstrated that deprivation of exogenous polyamines resulted in the developmental block of oogenesis and embryogensis in the C. elegans odc-1(pc13) loss-of-function mutant (13), which is consistent with the absolute requirement of polyamines for eukaryotic cell growth. Polyamine synthesis was also found to be essential for embryogenesis of other metazoa (41), and homozygous ODC- as well as AdoMetDC-deficient mouse embryos did not proceed beyond the blastocyst stage (42, 43). However, developmental blockage could not be overcome by uptake of polyamines from exogenous sources or parental animals, preventing studies on postembryonic effects. In contrast, C. elegans is a suitable metazoan model to investigate the effects of loss of polyamine synthesis on postembryonic developmental processes. In accordance with the polyamine auxotrophy reported for the odc-1(pc13) mutant, the C. elegans double mutants that lack a functional polyamine synthesis and the CATP-5 protein contained extremely altered polyamine levels. Particularly, the reduced spermidine concentrations were found to correlate with severe defects in several developmental parameters that were generally found to be less pronounced or even not seen in the single mutants with moderately changed polyamine pattern, such as a reduced number of progeny, retarded postembryonic development, small body size, and shortened adult life span. These data imply that polyamines are not only crucial for embryogenesis, but also function as important regulators of postembryonic processes.

Supplementary Material

Supplemental Data
fj.09-135889_index.html (772B, html)

Acknowledgments

Some C. elegans strains were obtained from the Caenorhabditis Genetics Center at the University of Minnesota (Minneapolis, MN, USA), which is funded by the U.S. National Institutes of Health National Center for Research Resources and by the National Bioresource Project, Tokyo Women’s Medical University School of Medicine. This work was supported by Deutsche Forschungsgemeinschaft (DFG; grant Lu 733/6) and is part of A.H.’s doctoral study at the Faculty of Biology, University of Muenster.

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