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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 - J. Cell Biol. (1997)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Nutrition, Harvard School of Public Health, Boston, Massachusetts 02115, USA.

ABSTRACT
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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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).
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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).

Mentions: PMA upregulates non–Tf-bound Fe uptake across the plasma membrane of K562 cells by a transcription-dependent mechanism (Akompong et al., 1995). Taking advantage of this observation, efforts were initiated to identify the elements involved in membrane Fe transport by functional expression in Xenopus oocytes. Fig. 1 A demonstrates that upon injection of mRNAs from control and PMA-treated K562 cells, 55Fe uptake by oocytes is stimulated two- and fourfold above background, respectively. Minimal transport activity is detected in control or water-injected cells. Nomizu et al. (1993) have reported that the Fe content of stage VI oocytes remains unchanged during subsequent maturation and fertilization, indicating that Fe uptake is limited with maternal stores providing sufficient Fe throughout early stages of development. Fractionation experiments confirmed the specific Fe transport by injected oocytes, since assimilated 55Fe was incorporated into cytosolic components with <5% of radioactivity associated with membranous components (not shown). Preliminary experiments also revealed that Fe uptake was time and temperature dependent (see below). Thus, expression of Fe transport activity by Xenopus oocytes programmed with K562 cell mRNA bears functional resemblance to the PMA-inducible, Tf-independent Fe carrier previously characterized for K562 cells (Inman and Wessling-Resnick, 1993).


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

Gutierrez JA, Yu J, Rivera S, Wessling-Resnick M - J. Cell Biol. (1997)

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).
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Related In: Results  -  Collection

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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).
Mentions: PMA upregulates non–Tf-bound Fe uptake across the plasma membrane of K562 cells by a transcription-dependent mechanism (Akompong et al., 1995). Taking advantage of this observation, efforts were initiated to identify the elements involved in membrane Fe transport by functional expression in Xenopus oocytes. Fig. 1 A demonstrates that upon injection of mRNAs from control and PMA-treated K562 cells, 55Fe uptake by oocytes is stimulated two- and fourfold above background, respectively. Minimal transport activity is detected in control or water-injected cells. Nomizu et al. (1993) have reported that the Fe content of stage VI oocytes remains unchanged during subsequent maturation and fertilization, indicating that Fe uptake is limited with maternal stores providing sufficient Fe throughout early stages of development. Fractionation experiments confirmed the specific Fe transport by injected oocytes, since assimilated 55Fe was incorporated into cytosolic components with <5% of radioactivity associated with membranous components (not shown). Preliminary experiments also revealed that Fe uptake was time and temperature dependent (see below). Thus, expression of Fe transport activity by Xenopus oocytes programmed with K562 cell mRNA bears functional resemblance to the PMA-inducible, Tf-independent Fe carrier previously characterized for K562 cells (Inman and Wessling-Resnick, 1993).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Nutrition, Harvard School of Public Health, Boston, Massachusetts 02115, USA.

ABSTRACT
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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