A surface glycoprotein indispensable for gamete fusion in the social amoeba Dictyostelium discoideum

Abstract

Sexual reproduction is essential for the maintenance of species in a wide variety of multicellular organisms, and even unicellular organisms that normally proliferate asexually possess a sexual cycle because of its contribution to increased genetic diversity. Information concerning the molecules involved in fertilization is accumulating for many species of the metazoan, plant, and fungal lineages, and the evolutionary consideration of sexual reproduction systems is now an interesting issue. Macrocyst formation in the social amoeba Dictyostelium discoideum is a sexual process in which cells become sexually mature under dark and submerged conditions and fuse with complementary mating-type cells. In the present study, we isolated D. discoideum insertional mutants defective in sexual cell fusion and identified the relevant gene, macA, which encodes a highly glycosylated, 2,041-amino-acid membrane protein (MacA). Although its overall similarity is restricted to proteins of unknown function within dictyostelids, it contains LamGL and discoidin domains, which are implicated in cell adhesion. The growth and development of macA-null mutants were indistinguishable from those of the parental strain. The overexpression of macA using the V18 promoter in a macA-null mutant completely restored its sexual defects. Although the macA gene encoded exactly the same protein in a complementary mating-type strain, it was expressed at a much lower level. These results suggest that MacA is indispensable for gamete interactions in D. discoideum, probably via cell adhesion. There is a possibility that it is controlled in a mating-type-dependent manner.

Fig 1

Generation of sexually defective mutants. (A) The genomic structure of the macA region for the parental KAX3 strain is shown on top. The thick blue and white bars indicate the exon of macA and neighboring genes, respectively. The triangle shows the vector insertion site in MCF8-5. For the other 3 mutants, substitutions to the vector sequences are shown by dotted lines. Red bars and arrows indicate the region used for the hybridization probe and the quantitative RT-PCR primers, respectively. Restriction sites are the following: B, BamHI; C, ClaI; E, EcoRI; H, HpaI; V, EcoRV; and X, XbaI. (B) Macrocyst (a, b, and c) and fruiting body formation (d, e, and f) of parental KAX3 (a and d), the macA-null mutant MCN1 (b and e), and the macA overexpression mutant in MCN1, MCR1 (c and f). For macrocyst formation, 100 cells were mixed with an equal number of V12 cells in 100 μl of BSS containing condensed K. aerogenes in a well of a 96-well plate and cultured for 4 days at 22°C. Bar, 100 μm. (C) Results of the cell fusion assays are shown. (D) Structure of the macA overexpression vector. The coding sequence of macA was inserted between the V18 promoter and the act8 terminator of pTMV18.

Fig 2

Expected structure of MacA. (A) The deduced amino acid sequence is shown. The yellow and green highlights indicate the signal peptide (SP) and transmembrane (TM) sequences, respectively. Possible N-glycosylation (pink) and O-glycosylation (red) sites are also highlighted. Blue and red underlines represent LamGL and F5/8C (discoidin [DS]) domains, respectively. O-glycosylation sites were predicted using the NetOGlyc 3.1 server (http://www.cbs.dtu.dk/services/netoglyc/). (B) The overall structure of MacA is shown schematically.

Fig 3

Detection of macA transcripts. (A) One hundred mg of poly(A)⁺ RNA prepared from FC cells of KAX3 was analyzed by Northern hybridization. The blot membrane was probed with the ³²P-labeled macA fragment shown in Fig. 1 (lane 1) or clone SLH805 from the Tsukuba cDNA project, representing a 3′-portion of mhcA (lane 2). (B) Quantitative RT-PCR amplification was performed for RNA prepared from fusion-competent (FC) or -incompetent cells cultured on a bacterial plate (ICP) and slug-stage cells of KAX3.

Fig 4

Conservation of macA in other mating-type strains of D. discoideum. (A) Genomic DNA was prepared from KAX3 (K), V12 (V), WS2162 (W), and AC4 (A) cells, digested with EcoRI, and subjected to Southern hybridization using a ³²P-labeled KAX3 macA fragment as a probe, which is shown by a red bar. (B) Amino acid alterations in WS2162 MacA. Due to an amino acid deletion, WS2162 MacA is 2,040 amino acids long. (C) Approximate positions of the deletion (pink arrow) and substitutions (black arrows) in WS2162 are mapped. (D) The similarity of AC4 MacA for every 120 amino acids is shown.

Fig 5

Differential detection of macA expression with mating type. (A) The peptide hits from proteome analysis were plotted for MacA (a), Nad7 (b), and MhcA (c). (B) A portion of macA cDNA was amplified for FC cells of KAX3 (K), V12 (V), and WS2162 (W) using the primer set shown in Table S1 in the supplemental material.