The P450 oxidoreductase, RedA, controls development beyond the mound stage in Dictyostelium discoideum

Abstract

Background: NADPH-cytochrome-P450 oxidoreductase (CPR) is a ubiquitous enzyme that belongs to a family of diflavin oxidoreductases and is required for activity of the microsomal cytochrome-P450 monooxygenase system. CPR gene-disruption experiments have demonstrated that absence of this enzyme causes developmental defects both in mouse and insect.

Results: Annotation of the sequenced genome of D. discoideum revealed the presence of three genes (redA, redB and redC) that encode putative members of the diflavin oxidoreductase protein family. redA transcripts are present during growth and early development but then decline, reaching undetectable levels after the mound stage. redB transcripts are present in the same levels during growth and development while redC expression was detected only in vegetative growing cells. We isolated a mutant strain of Dictyostelium discoideum following restriction enzyme-mediated integration (REMI) mutagenesis in which redA was disrupted. This mutant develops only to the mound stage and accumulates a bright yellow pigment. The mound-arrest phenotype is cell-autonomous suggesting that the defect occurs within the cells rather than in intercellular signaling.

Conclusion: The developmental arrest due to disruption of redA implicates CPR in the metabolism of compounds that control cell differentiation.

Figure 1

Alignment of CPR aminoacid sequences. Comparison of Dictyostelium discoideum (Dd) NADPH cytochrome P450 oxidoreductase (RedA) aminoacid sequence with orthologs from Homo sapiens (Hs, P16435), Rattus norvegicus (Rn, P00388), Drosophila melanogaster (Dm, CAA63639) and Saccharomyces cerevisiae (Sc, P16603) using CLUSTAL W program. Identical and conserved amino acids are denoted with (*) and (:), respectively. Semi-conservative changes are indicated with a single dot. Binding domains for FMN, FAD and NADPH are indicated. Closed circles indicate highly conserved aromatic amino-acid residues that are particularly important in flavin binding. Open triangle points residues involved in NADPH discrimination. The ~56 aminoacid and the ~20 aminoacid hydrophobic segments at the N-terminal are indicated by dashed line in human CPR and dotted line in Dictyostelium CPR, respectively.

Figure 2

Expression of redA during growth and development of wild type AX4 cells. Exponentially growing AX4 cells (Veg) were starved on filter pads and harvested at the indicated times (h) after starvation. Identical Northern blots of total RNA samples were probed with redA, IG7 and csaA cDNAs as indicated. IG7 transcript is expressed at similar levels throughout the D. discoideum development [59].

Figure 3

Transcriptional profile of redA, redB, redC and ecmA. Exponentially growing AX4 (A) and redA^- (B) cells were starved on filter pads and harvested at the indicated times (h) after starvation. Transcript levels for redA, redB and redC genes are relative to 0 h cells. Fold change for ecmA are relative to transcript levels detected at 16 h. Error bars represent the standard deviation from two independent experiments where qPCR assays were performed in triplicate.

Figure 4

Disruption of redA impairs development at mound stage. (A) Exponentially growing AX4 wild type cells and the mutants redA^- and redA-KO were starved on filter pads and photographed at the indicated times (h) after starvation. (B) AX4 fruiting bodies and redA^- yellow mounds after 48 hours starvation on filter pads are shown at lower (left) and at 5× higher magnification (right).

Figure 5

Disruption of redA results in cells lacking redA transcript. (A) Total RNA was prepared from exponentially growing AX4 cells (AX4) and from two independent clones of the mutants redA^- and redA-KO. Identical Northern blots were probed with redA and IG7 cDNAs as indicated. (B) Exponentially growing redA^- cells (Veg) were starved on filter pads and harvested at the indicated times (h) after starvation. Identical Northern blots of total RNA samples were probed with redA, IG7 and csaA cDNAs as indicated.

Figure 6

AX4 cells do not rescue redA^- phenotype. Exponentially growing redA^- and AX4 wild type cells were starved on filter pads mixed at the indicated proportions. At the indicated times (h) after starvation cells were photographed.

Figure 7

UV and visible spectra of the chloroform extracts from AX4 and redA^-. Chloroform extracts of AX4 and redA^- cells collected after 48 hours starvation were analyzed using a UV/Visible spectrophotometer. The arrow points the absorption peak at 400 nm observed in the redA^- cell extract.