ADI1, a methionine salvage pathway enzyme, is required for Drosophila fecundity

Abstract

Background: Methionine, an essential amino acid, is required for protein synthesis and normal cell metabolism. The transmethylation pathway and methionine salvage pathway (MTA cycle) are two major pathways regulating methionine metabolism. Recently, methionine has been reported to play a key role in Drosophila fecundity.

Results: Here, we revealed that the MTA cycle plays a crucial role in Drosophila fecundity using the mutant of aci-reductone dioxygenase 1 (DADI1), an enzyme in the MTA cycle. In dietary restriction condition, the egg production of adi1 mutant flies was reduced compared to that of control flies. This fecundity defect in mutant flies was rescued by reintroduction of Dadi1 gene. Moreover, a functional homolog of human ADI1 also recovered the reproduction defect, in which the enzymatic activity of human ADI1 is required for normal fecundity. Importantly, methionine supply rescued the fecundity defect in Dadi1 mutant flies. The detailed analysis of Dadi1 mutant ovaries revealed a dramatic change in the levels of methionine metabolism. In addition, we found that three compounds namely, methionine, SAM and Methionine sulfoxide, respectively, may be required for normal fecundity.

Conclusions: In summary, these results suggest that ADI1, an MTA cycle enzyme, affects fly fecundity through the regulation of methionine metabolism.

Figure 1

Isolation ofDadi1 alleles. (A) Schematic diagram of the methionine salvage and recycling pathways, which is generated based on metabolism pathways in the Kyoto Encyclopedia Genes and Genomes (KEGG) database. The Main enzyme studied here is highlighted with yellow. (B) A map of genomic DNA displays the Dadi1 (CG32068) locus. The DADI1^d01129 P-element insertion line (black arrow) contains a p-element at 48 bp upstream of the DADI1 start codon (red arrow). Three DADI1 mutant alleles (Dadi1⁷, Dadi1⁹ and Dadi1⁷⁴) were produced with alterations in the Dadi1 coding region. (C) Immunoblotting of DADI1 in several developmental stages. Embryos were collected after egg deposition. (D) The protein levels of DADI1 were reduced in Dadi1⁷⁴ heterozygote strain (Dadi1⁷⁴/TM3Ser). Western blot analysis of DADI1 protein levels of Dadi1⁷/ Dadi1⁹ trans-heterozygous alleles and Dadi1⁷⁴ homozygous mutant indicated that they were protein null alleles.

Figure 2

DADI1 is required forDrosophilafecundity. (A) Eggs of control and Dadi1 mutant flies were enumerated and normalized to control flies under the dietary restriction condition. (B) Western blot analysis of DADI1 protein levels in the adult lysate of control and DADI1 transgenic lines, which expressed in Dadi1 null mutant. (C) The fecundity defect was rescued in Dadi1 mutant alleles expressing UAS-DADI1 driven by Actin-Gal4. *P < 0.05, **P < 0.01.

Figure 3

Enzymatic activity of ADI1 is essential for regulating egg production. (A) Protein sequence alignment is shown in the Cupin domain of hADI1 and DADI1. The identity and similarity of Cupin domain is 68% and 84%, respectively. The glutamic acid site is required the ARD/ARD’ family to execute enzymatic function (red star). (B) The protein expression levels of human wild-type and E94A mutant transgenic lines were observed in the Dadi1 null mutant background by hADI1 antibody. (C) Expressing human ADI1 by Actin-Gal4 can rescue the egg production defect in Dadi1 mutant alleles. (D) The fecundity was not rescued by diverse expressing enzyme dead mutant (E94A) transgenic lines. *P < 0.05, **P < 0.01 and N.S., no significance.

Figure 4

Methionine supplementation rescued the fecundity defect inDadi1mutant alleles. The fecundity of Dadi1 null mutants was recovered to the level of control flies after adding back met but not trp. *P < 0.05, **P < 0.01 and N.S., no significance.

Figure 5

The metabolites of MTA cycle are affected byDADI1. (A) Four metabolites, Methionine, SAM, MTA and Methionine sulfoxide, were observed to be rescued in Dadi1 mutant ovaries by methionine addition. (B) Under the restricted diet condition, the metabolites of the methyl cycle and MTA cycle were dramatically changed in Dadi1 mutant ovaries. The raw data of metabolites are displayed below the picture. The results represent the means ± SD of three experiments. *P < 0.05, **P < 0.01 and N.S. indicates no significance.