Ferroportin: lack of evidence for multimers

Schimanski LM, Drakesmith H, Talbott C, Horne K, James JR, Davis SJ, Sweetland E, Bastin J, Cowley D, Townsend AR. (2008), Blood Cells Mol Dis. 40, 360-9

Abstract

Ferroportin is a multi-transmembrane glycoprotein that mediates iron export from cells. Mutations in ferroportin are linked to type IV hemochromatosis, a dominantly inherited disorder of iron metabolism. Multimers of ferroportin, whose existence may relate to the dominant inheritance pattern of disease, have been detected in some studies but not others. We looked for evidence of multimerization in several different types of experiment. We assayed the maturation of mutant and wild-type ferroportin and found that loss-of-function mutants had a reduced half-life but did not alter the stability of coexpressed wild-type. Using bioluminescence resonance energy transfer analysis, we tested how mature wild-type ferroportin behaved in intact live cell membranes. Ferroportin-ferroportin interactions gave the very low acceptor/donor ratio-independent energy transfer levels characteristic of random protein-protein interactions, consistent with ferroportin behaving as a monomer. Consistent with these experiments, we were unable to detect a dominant negative functional effect of mutant ferroportin on wild-type, even when expression of wild-type protein was titrated to low levels. These data suggest that dominantly inherited ferroportin disease does not result from the direct action of a mutated protein inhibiting a wild-type protein within multimers. We propose other possible mechanisms of disease.

Key figure: In bioluminescence resonance energy transfer assays, ferroportin exhibits monomeric behaviour

(A) 293T cells were transfected with constant overall amounts but varying ratios of constructs encoding wild-type FPN.GFP² and FPN.Luc (filled circles), β₂AR.GFP² and FPN.Luc (open circles), or GABA_βR2.GFP²and GABA_βR1.Luc (filled triangles) as described [21]. BRET measurements of FPN with FPN yielded values similar to those of FPN with β₂AR; FPN and β₂AR are assumed not to interact and behave as independent monomers. The FPN data fits well to a constant value (dotted line at ∼ 0.14), further indicative of monomeric behaviour. In contrast the heterodimerizing multi-transmembrane proteins GABA_βR1 and GABA_βR2 gave higher BRET values that exhibit hyperbolic dependence on the acceptor/donor ratio. (B) 293T cells transfected with constant overall amounts but varying ratios of constructs encoding wild-type FPN.GFP² and FPN.Luc were exposed to 0.5 μM hepcidin either overnight (open triangles) or for 20 min (open squares) immediately preceding the BRET analysis. Hepcidin did not induce the higher BRET values that if observed would have suggested oligomerization BP = BRET pair.