Fig. 1

Hrg1 is duplicated in the zebrafish genome.

(A) Two hrg1 homologs exist in zebrafish genome: hrg1a (slc48a1b) on chromosome 6 and hrg1b (slc48a1a) on chromosome 23. (B) Phylogenetic analysis of zebrafish Hrg1a and Hrg1b with CeHRG-1, CeHRG-4, MmHrg1, and Human Hrg1 (HsHrg1). Sequences were aligned using ClustalW and a phylogenetic tree was generated using the Neighbor-Joining method in MEGA5. (C) Multiple sequence alignment of zebrafish Hrg1a and Hrg1b with orthologs CeHRG-1, Cehrg-4, MmHrg1, and Human Hrg1 (HsHrg1). Asterisk, conserved histidine; black box, putative transmembrane domains; YXXxØ, C-terminal tyrosine sorting motif; D/EXXxLL, di-leucine sorting motif. (D) qRT-PCR of hrg1a and hrg1b in zebrafish embryos at different stages. 50 embryos before 24hpf were collected as one cohort. For embryos later than 24hpf, 30 were pooled as one cohort. 3 cohorts of each stage were harvested for total RNA extraction. Expression level was normalized to ß-actin. (E) Lateral view of hrg1a and hrg1b expression by WISH at 2dpf. Anterior is to the left. Scale bar: 200μm. (F) Confocal microscopy depicting subcellular localization of Hrg1a and Hrg1b with C-terminally tagged fluorescent proteins in transfected HEK-293 cells. Lamp1 is a marker for lysosomal compartments. Scale bar: 10 μm. (G) Yeast growth assay showing that zebrafish Hrg1a and Hrg1b are able to mediate heme transport in yeast. The hem1Δ strains transformed with empty vector pYes-DEST52, CeHRG-4, CeHRG-1, Hrg1a and Hrg1b were cultivated overnight and spotted in serial dilutions on SC plates supplemented with 250μM ALA and indicated concentrations of heme.