SLC25 Family Member Genetic Interactions Identify a Role for HEM25 in Yeast Electron Transport Chain Stability

Abstract

The SLC25 family member SLC25A38 (Hem25 in yeast) was recently identified as a mitochondrial glycine transporter that provides substrate to initiate heme/hemoglobin synthesis. Mutations in the human SLC25A38 gene cause congenital sideroblastic anemia. The full extent to which SLC25 family members coregulate heme synthesis with other mitochondrial functions is not clear. In this study, we surveyed 29 nonessential SLC25 family members in Saccharomyces cerevisiae for their ability to support growth in the presence and absence of HEM25 Six SLC25 family members were identified that were required for growth or for heme synthesis in cells lacking Hem25 function. Importantly, we determined that loss of function of the SLC25 family member Flx1, which imports FAD into mitochondria, together with loss of function of Hem25, resulted in inability to grow on media that required yeast cells to supply energy using mitochondrial respiration. We report that specific components of complexes of the electron transport chain are decreased in the absence of Flx1 and Hem25 function. In addition, we show that mitochondria from flx1Δ hem25Δ cells contain uncharacterized Cox2-containing high molecular weight aggregates. The functions of Flx1 and Hem25 provide a facile explanation for the decrease in heme level, and in specific electron transport chain complex components.

Keywords: SLC25 protein family; electron transport chain; glycine import; heme; mitochondria.

Figure 1

Mitochondrial glycine is used to synthesize heme and produce one-carbon units. (A) Heme biosynthesis pathway. (B) Mitochondrial glycine can be used to synthesize heme or catabolized by the GCV into nitrogen and one-carbon units. Hem25, the yeast homolog of human SLC25A38, is a mitochondrial glycine importer, and Lpd1 is a subunit of the yeast GCV. ALAS, aminolevulinic acid synthase; ALAD, aminolevulinic acid dehydratase; PBGD, porphobilinogen deaminase; UROS, uropoporphyrinogen III synthase; UROD, uroporphyrinogen III decarboxylase; CPOX, coproporphyrinogen oxidase; PPO, protoporphyrinogen oxidase; FECH, ferrochelatase; GCV, glycine cleavage complex.

Figure 2

Growth rates on glycine as sole nitrogen source for selected members of the SLC25 family in combination with HEM25 deficiency. Cells of the indicated genotypes were grown overnight on SD-Ura medium containing 1 g/liter of ammonium sulfate at 30°, washed twice and inoculated at OD_{600 nm} 0.1 in SD-Ura medium containing 30 g/liter of glycine. Cells were cultivated at 30°, and growth was monitored spectrophotometrically. Differences between single deletion strains and double deletion strains were determined using ANOVA test with randomized factors. At least three independent experiments were done to calculate the p values, mean, and SEM. The numbers represented in the graph are growth rates calculated over a period of 4 d.

Figure 3

Heme content for selected members of the SLC25 family in combination with HEM25 deficiency. Cells of the indicated genotypes were grown at OD_{600 nm} 0.6–1 in SD-Ura. Cells were harvested and processed for heme determination. Differences between single deletion strains and double deletion strains were determined using ANOVA test with randomized factors. At least three independent experiments were done to calculate the p values, mean, and SEM. Values are normalized to wild type (WT). WT heme level was 28.9 ± 4.2 fmol/μg of protein.

Figure 4

Glycine and 5-Ala alleviate the growth defect of a subset of SLC25 family members upon inactivation of the mitochondrial glycine importer HEM25. Yeast strains of the indicated genotypes were grown to early stationary phase in SC-Ura medium supplemented with 5 mM glycine and 0.38 mM 5-Ala to keep the double mutant cells growing without impediment. Cells were washed and resuspended in sterilized water to OD_{600 nm} 0.4, then serially diluted (1:10) and spotted on SD-Ura solid media containing dextrose as carbon source. Plates were imaged after 3 d incubation at 30°.

Figure 5

Growth rescue by glycine and 5-Ala on nonfermentable medium for cells lacking specific SLC25 family members in concert with HEM25. Yeast strains of the indicated genotypes were grown to early stationary phase in SC-Ura medium supplemented with 5 mM glycine and 0.38 mM 5-Ala to keep the double mutant cells growing without impediment. Cells were washed and resuspended in sterilized water to OD_{600 nm} 0.4, then serially diluted (1:10) and spotted on SD-Ura solid media containing lactate as carbon source. Plates were imaged after 7 d incubation at 30°.

Figure 6

Specific components of the electron transport chain are significantly decreased in hem25Δ cells lacking the FAD importer FLX1. Cells of the indicated genotypes were grown to an OD of 1.0 in defined medium with raffinose. Cells were then transferred and grown in lactate for 5 hr. Cells were harvested and mitochondrial fractions were prepared and analyzed by western blotting using antibodies specific for (A) Ndi1, (B) Sdh1 and Sdh2, (C) Cor2 and Cyt1, (D) Cox2 and Cox4, and (E) F1α and F1β. Three independent segregant strains of flx1Δ and flx1Δ hem25Δ were used. The figure shown is representative of three independent analyses. Pixel intensity was measured and calculated by using Odyssey Software. Numbers under the lanes represent the mean of protein abundance normalized by the loading control Por1 and then to WT. The mean and the SEM were calculated from three independent western blot analyses. The significance of the differences on protein levels observed between the strains was calculated using ANOVA test with randomized factors (Table S3 in File S1). The deduced protein molecular weight (MW) of the bands revealed by western blotting was consistent with their MW: Ndi1 (57 kDa); Sdh1 (69 kDa) and Sdh2 (30 kDa); Cor2 (40 kDa) and Cyt1 (34 kDa); Cox2 (28 kDa) and Cox4 (17 kDa); F1α (58 kDa) and F1β (55 kDa).

Figure 7

High molecular weight aggregates are present in mitochondria from flx1Δ hem25Δ cells. Cells of the indicated genotypes were grown to an OD of 1.0 in defined medium with raffinose. Cells were then transferred and grown in lactate for 5 hr. Cells were harvested, mitochondria were isolated and mitochondrial protein complexes were solubilized with digitonin. Protein complexes were resolved by BN-PAGE, transferred into PVDF and analyzed by western blotting using antibodies specific for Cox2 (upper and middle panels; they are identical, middle panel was a longer exposure) or F1α (lower panel). Asterisk denotes the position of high molecular weight aggregates.