ABC Transporters in Dictyostelium discoideum Development

Abstract

ATP-binding cassette (ABC) transporters can translocate a broad spectrum of molecules across the cell membrane including physiological cargo and toxins. ABC transporters are known for the role they play in resistance towards anticancer agents in chemotherapy of cancer patients. There are 68 ABC transporters annotated in the genome of the social amoeba Dictyostelium discoideum. We have characterized more than half of these ABC transporters through a systematic study of mutations in their genes. We have analyzed morphological and transcriptional phenotypes for these mutants during growth and development and found that most of the mutants exhibited rather subtle phenotypes. A few of the genes may share physiological functions, as reflected in their transcriptional phenotypes. Since most of the abc-transporter mutants showed subtle morphological phenotypes, we utilized these transcriptional phenotypes to identify genes that are important for development by looking for transcripts whose abundance was unperturbed in most of the mutants. We found a set of 668 genes that includes many validated D. discoideum developmental genes. We have also found that abcG6 and abcG18 may have potential roles in intercellular signaling during terminal differentiation of spores and stalks.

The ABC transporter, AbcB3, mediates cAMP export in D. discoideum development

“…Multidrug resistance caused by ABC transporters has been an area of focused research and ABC transporter dysfunction causes 17 known genetic disorders (15). Our previous work (18) and this study provide a framework for studying ABC transporter function in D. discoideum not only in the context of amoeba biology but also in the context of human biology and disease.…”

“…As an alternative approach, we created abcB3 -RNAi strains. During development, abcB3 expression increases at 4 hours and peaks by 8 hours (18, 29), so we drove expression of the abcB3 shRNA using the tgrB1 element of the bidirectional tgrB1/C1 promoter (30, 31). Since tgrB1 expression begins at the onset of development, peaks at 4 hours, and remains high until 8 hours, we reasoned that the construct would allow for high levels of shRNA to be present when abcB3 expression increases and peaks, but not during growth.…”

“…Total RNA was extracted according to the manufacturer’s recommended protocol. RNA was treated with DNAse as described (30), cDNA prepared using Superscript II reverse transcriptase as described (29), and quantitative reverse transcriptase PCR (qRT-PCR) performed with oligonucleotides specific to each abc gene as described (18). Two independent incubation experiments with three technical replicates of the qRT-PCR were performed.…”

“…In D. discoideum there are 68 abc genes, which have been classified into 8 groups ( abcA through abcH ) based on sequence and predicted structural features (14), but their functions are largely unknown. We recently characterized the transcriptional and morphological phenotypes of 35 abc mutants during development and found that most mutants exhibited subtle phenotypes, suggesting that many of the ABC transporters could have overlapping functions (18). We hypothesized that cAMP exporters may be among the uncharacterized abc genes.…”

Altered N-glycosylation modulates TgrB1- and TgrC1-mediated development but not allorecognition in Dictyostelium

“…Cells were grown in shaking suspension in HL5 medium supplemented with 50 U/ml penicillin and 50 µg/ml streptomycin at 22°C (Miranda et al, 2013). In drug-resistant strains, 5 µg/ml Blasticidin-S or 10 µg/ml G418 was added as necessary.…”