Gutierrez JA et al. (1997), Functional expression cloning and characterizat...

XB-ART-15713

J Cell Biol 1997 Nov 17;1394:895-905. doi: 10.1083/jcb.139.4.895.

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Functional expression cloning and characterization of SFT, a stimulator of Fe transport.

Gutierrez JA , Yu J , Rivera S , Wessling-Resnick M .

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A stimulator of Fe transport (SFT) was identified by functional expression cloning in Xenopus oocytes. SFT-mediated transport has properties defined for transferrin-independent Fe uptake, but its cytolocalization in recycling endosomes and the observed stimulation of transferrin-bound Fe assimilation indicate a key role in intracellular Fe membrane transport as well. SFT has six predicted transmembranous domains and a functionally important RExxE motif that resembles domains involved in yeast Fe transport and Fe-binding by ferritin L-chains. The observation that SFT oligomerizes, along with other structural and mechanistic features, suggests it may be a member of either the ATP-binding cassette or cation diffusion facilitator families. The 3' untranslated region of SFT contains a translation inhibitory element and inhibition of SFT expression in Xenopus oocytes was found to be relieved by coinjection of transcripts from other defined cDNAs that are also described in this report. SFT is the first component of the mammalian Fe membrane transport machinery to be identified.

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Species referenced: Xenopus laevis
Genes referenced: tbx2 tf ube2d1

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	Figure 1. Iron transport in Xenopus oocytes. (A) Fe transport was measured for oocytes injected with water or 20 ng mRNAs from control or PMA-stimulated K562 cells. Shown are the average values of 55Fe assimilated over 2 h ± SE (n = 6 oocytes). (B) Fe transport was measured as in A for oocytes microinjected with water or 4 ng of the following: full-length SFT cRNA + pool of complementing cRNAs (15 oocytes), full-length SFT cRNA alone (15 oocytes), or SFT-ORF cRNA, which contains the ORF (17 oocytes). Data are the average values ± SE. (C) Time course of Fe assimilation by oocytes microinjected with 4 ng of SFT-ORF cRNA. 55Fe uptake was determined as a function of time; background levels measured for water- injected oocytes were subtracted to obtain specific transport measurements (mean ± SE; n = 15). (D) Fe transport was measured for oocytes injected with water or 4 ng of wild-type and E→ A mutant SFT-ORF cRNA. The latter mutant contains alanines in place of the key glutamic acid residues in the RExxE motif present in SFT (Glu83 and Glu86) that appears functionally related to a domain important for high affinity Fe transport in yeast (Stearman et al., 1996). Data are the average values ± SE (n = 12).
	Figure 2. Relative size and abundance of SFT transcripts. (A) Total RNA from control or PMA-treated K562 cells was probed with 32P-labeled SFT cDNA under high stringency hybridization conditions. Locations of 28S and 18S ribosomal bands are depicted by left arrows and the approximate molecular sizes for the two transcripts are shown on the right. Equal RNA loading was determined by hybridization with 32P-labeled cDNA for 36B4 ribosomal protein. (B) A Northern blot containing 2.5 μg poly(A) RNA from several human tissues was probed with 32P-labeled SFT cDNA. Molecular sizes are depicted on the left.
	Figure 4. Hypothetical model for SFT membrane structure. (A) Hydropathy plot of SFT amino acid sequence by Kyte-Doolittle analysis (Kyte and Doolittle, 1982). (B) Proposed membrane orientation for SFT from amino acid sequence shown in Fig. 3.
	Figure 5. In vitro translation of SFT cRNA in the presence of microsomes. Rabbit reticulocyte lysate was programmed with water (lane A), 100 ng SFT-ORF cRNA (lane B), or 100 ng full-length SFT cRNA (lane C). [35S]Methionine-labeled products were electrophoresed on a 10% SDS–polyacrylamide gel; a fluorograph of the dried gel is shown. SFT is denoted by the right arrow; molecular weight standards (left) were bovine serum albumin (66 kD), glyceraldehyde 3–phosphate dehydrogenase (36 kD), carbonic anhydrase (29 kD), and soybean trypsin inhibitor (20 kD).
	Figure 6. GFP chimera of SFT associates with recycling endosomes. To visualize endocytic compartments, HeLa cells transiently expressing GFP-SFT were incubated with Texas red–Tf as described in Materials and Methods. The upper image is Texas red–Tf fluorescence and overlap with GFP-SFT (bottom) is indicated with arrows. To verify that the presence of GFP at SFT's COOH terminus does not interfere with its normal cellular localization, a chimera of SFT with two NH2-terminal HA tags was also constructed and found to localize to the same domain (not shown).
	Figure 7. SFT stimulates assimilation of Fe from Tf. The accumulation of 55Fe from Tf was measured for HeLa cells stably expressing the SFT-GFP chimera (•) as well as control cells (○) as described in Materials and Methods. The amount of cell-associated radioactivity determined for duplicate samples ± SE is shown as a function of time.
	Figure 8. Meiotic maturation of oocytes with progesterone promotes expression of SFT activity. Fe transport was measured as in Fig. 1 except that some oocytes were treated with 1 μM progesterone 24 h after injection (solid bars). Transport of 55Fe was normalized by subtracting measurements made for water-injected oocytes for each condition. Shown are results from a single experiment and are representative of those obtained on three separate occasions; data are the average values ± SE (n = 15 oocytes).

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Akompong, Phorbol esters stimulate non-transferrin iron uptake by K562 cells. 1995, Pubmed