A Putative Ariadne-Like Ubiquitin Ligase Is Required for Dictyostelium discoideum Development

The RBR family of ubiquitin ligases is a large and complex family with members present in animals, plants, fungi, and protists (17). RBR genes have been implicated in cancer and neurodegenerative diseases (e.g., parkin) (17-19, 21). Despite significant advances, much of the potential diversity in RBR protein function remains to be studied. Particularly interesting may be members of the Ariadne subfamily, since this subfamily appears to be the most ancient (1, 3, 17). In this report, we identify a putative Ariadne ubiquitin ligase, RbrA, in Dictyostelium. Dictyostelium discoideum Ax4 and DH1 were used as wild-type and parental strains. All strains were grown in HL5 with glucose (Qbiogene, Carlsbad, CA) or with Klebsiella aerogenes on agar plates as described previously (12). Multicellular development, preparation of conditioned starvation medium, phototaxis studies, Northern blot analysis, and preparation of cDNA clones were done as previously described (12, 16, 20). Restriction enzyme-mediated integration (REMI) was carried out as described previously (14). An rbrA mutant was found in a screen of REMI transformants as cells whose development is blocked at the finger/slug stage. rbrA mutant cells formed ripples by 2 h of development, which was earlier than wild type (Fig. ​(Fig.1);1); however, development then slowed compared to wild-type cells and fruiting bodies never formed. In mixtures of rbrA− (90%) and wild-type (10%) cells, early rippling was observed, but nearly all structures produced by the chimeras formed fruiting bodies (Fig. ​(Fig.1).1). The addition of wild-type conditioned medium to rbrA− cells did not cause a similar phenotype rescue. Thus, a small number of wild-type cells appeared to rescue the morphological developmental defect, and the rescue appeared to require cell-cell proximity. FIG. 1. Developmental phenotype of rbrA mutant cells. (A) Wild-type cells, rbrA mutant cells generated by REMI mutagenesis or rbrA mutant cells generated by replacement of a large fragment of the rbrA coding region with a blasticidin resistance cassette and ... When developed on filters, we observed that slugs formed from rbrA− cells were capable of migrating at least short distances. However, slugs were unable to phototax when developed on agarose in a dark chamber with a directional light source (Fig. ​(Fig.1B).1B). Slugs that formed from a mixture of rbrA− and wild-type (10%) cells migrated directionally toward the light source and traveled nearly as far as wild type (Fig. ​(Fig.1B).1B). These are likely chimeric slugs because at least 50% of the slugs that formed showed positive phototaxis, and other experiments showed that chimeric slugs do form (see Fig. ​Fig.44). FIG. 4. Cell sorting is altered in the rbrA mutant. (A) Wild-type and rbrA− cells were transformed with cell-type-specific GFP expression plasmids PsA-ubi-s65tGFP or 63-ubi-s65tGFP, which contained the GFP cDNA expressed by the prestalk-specific promoter ... Genomic rbrA DNA was cloned by digestion with BglII to generate plasmid pM10B (Fig. ​(Fig.2A).2A). The pM10B plasmid was linearized with BglII and used to transform a wild-type host strain (DH1). The resulting gene disruption by homologous recombination successfully recapitulated the original rbrA mutant phenotype. A second rbrA mutant was constructed by using a gene targeting plasmid, pRbrBsr, to replace a portion of the rbrA gene with a blasticidin-resistance cassette (Fig. ​(Fig.2B).2B). This rbrA− strain demonstrated a mutant phenotype nearly identical to the original rbrA mutant (Fig. ​(Fig.1).1). Expression of the rbrA cDNA complemented the defective developmental phenotype of rbrA− cells (Fig. ​(Fig.2C).2C). A 2.2-kb rbrA mRNA was present in vegetatively growing cells (0 h), increased in level at 9 h (Fig. ​(Fig.2D),2D), and remained at the 9-h level throughout the remainder of development (data not shown). rbrA mRNA was not present in rbrA− cells (Fig. ​(Fig.2D2D). FIG.2. rbrA gene. (A) Genomic map and REMI-generated mutation. REMI was carried out by electroporating EcoRI-linearized DIV2 (the mutagenic plasmid carrying the Dictyostelium pyr5-6 gene) into DH1 (pyr5-6−) cells along with the restriction enzyme MunI ... rbrA genomic and cDNA clones revealed a single long open reading frame interrupted by two introns (Fig. ​(Fig.2A).2A). The deduced amino acid sequence predicts a protein of 520 residues with a molecular mass of 61 kDa (Fig. ​(Fig.2E).2E). RbrA has strong sequence similarity to the Ariadne subfamily of the RBR family of E3 ubiquitin ligases (17). The characteristic series of acid-rich, RBR (for “RING, between rings, RING”), and coiled-coil/leucine rich motifs found in Ariadne subfamily members are present in RbrA (Fig. ​(Fig.2E).2E). RbrA has 34% identity (54% similarity) to human HHARI. These data suggest that rbrA encodes an E3 ligase. To investigate the function of RbrA, we examined the expression of developmentally regulated genes. Transcript levels for the prestalk-cell-specific ecmA gene (23) were lower in rbrA− cells compared to wild-type cells (Fig. ​(Fig.3).3). The prespore-specific pspD (25) was regulated temporally in developing rbrA− cells as in wild-type cells, but at much higher than wild-type levels (Fig. ​(Fig.3).3). In addition, transcript levels for gpaD (required for spore formation) (10) were elevated and gpaE (tip formation) (11) levels were lower during rbrA− cell development (Fig. ​(Fig.33). FIG. 3. Gene expression in rbrA− cells. Total RNA was prepared from wild-type cells (Ax4) at the indicated stages of development (in hours). RNA (5 μg) was resolved by electrophoresis through 1.2% agarose-formaldehyde gels, blotted onto nylon ... To investigate cell-type proportioning and cell sorting, rbrA− and parental cells were transformed with cell-type-specific green fluorescent protein (GFP) expression plasmids (5). When the ecmAO promoter was used to express GFP in wild-type cells, fluorescence appeared in the anterior prestalk region, as expected (6, 13) (Fig. ​(Fig.4A);4A); however, rbrA− slugs showed a very low level of fluorescence throughout the slug. GFP expressed from the pspA promoter marked the posterior prespore region in wild-type slugs, whereas the entire rbrA− slug was marked (Fig. ​(Fig.4A).4A). Thus, prespore cells were prevalent and appeared to dominate the entire rbrA− slug. To determine whether the initial cell-autonomous differentiation was altered in rbrA− cells (2, 8, 22), cells were starved at low cell density and the numbers of CP2-positive prestalk cells and SP70-positive prespore cells were counted (9). The percentage of CP2- and SP70-positive wild-type cells was similar to what we previously observed (4, 24). There was essentially no difference in the initial differentiation of rbrA− cells compared to wild-type cells (Table ​(Table11). TABLE 1. Cell-type proportioning is altered in rbrA− slugsa To determine the percentage of prestalk and prespore cells in slugs, wild-type and rbrA− slugs were dissociated into single cells, fixed, and stained for prespore (SP70) or prestalk (CP2) markers (7). The percentage of CP2- and SP70-positive wild-type cells was similar to what we previously observed (4). rbrA− slugs had a percentage of SP70-positive prespore cells that was similar to that of the wild type but had threefold-fewer CP2-positive prestalk cells than did the wild type (Table ​(Table1).1). Together, the data suggest that the initial cell-autonomous differentiation of rbrA− cells into CP2-positive prestalk cells and SP70-positive prespore cells is normal and that rbrA− slugs contain an abnormally low number of CP2-positive prestalk cells. To determine the effect of wild-type cells on rbrA− cell-fate choice, mixtures of wild-type and rbrA− cells were prepared in which one of the strains contained the GFP gene driven by the constitutively expressed actin-15 promoter (15). Wild-type and rbrA− cells were mixed in various proportions and developed on filters. Fluorescence imaging of chimeric slugs indicated that the wild-type cells preferentially located to the tip region, which is occupied by prestalk cells in pure wild-type slugs (Fig. ​(Fig.4B).4B). The rbrA− cells preferentially located to the posterior of the slug, which normally is populated with prespore cells. To investigate the ability of rbrA− cells to form spores during chimeric development, spore assays were carried out on fruiting bodies produced by chimeras (6). Experiments with chimeras containing 3 to 40% rbrA− cells produced less than 1% detergent-resistant spores with the rbrA− phenotype, suggesting that rbrA− spores were sensitive to detergent treatment. We therefore carried out additional experiments in which spores were plated before and after detergent treatment (Table ​(Table2).2). The results showed that rbrA− cells did not efficiently survive the terminal differentiation stages. TABLE 2. rbrA− cells do not efficiently produce viable sporesa The predicted amino acid sequence of Dictyostelium RbrA suggests that it is a member of the Ariadne subfamily of ubiquitin ligases. RbrA appears to be required for normal cell-type proportioning and cell sorting during multicellular development. Prestalk cell numbers are reduced in rbrA− slugs and these prestalk cells do not localize to the tip of slugs. Development terminates at the finger or slug stage, and these slugs do not phototax, possibly because the tip region does not properly form. In addition to being necessary for a normal percentage of prestalk cells and the organization of the slug, RbrA is also necessary for spore cell viability. RbrA thus affects multiple processes such as prestalk cell differentiation, pattern formation, and spore cell maturation.