Abstract
Slc11a1 (formerly Nramp1) is a proton/divalent cation transporter that regulates cation homeostasis in macrophages. Slc11a2 mediates divalent cation uptake via the gut and delivery into cells. The mode of action of the two transporters remains controversial. Heterologous expression in frog oocytes shows Slc11a2 is a symporter, whereas Slc11a1 is an antiporter fluxing divalent cations against the proton gradient. This explains why Slc11a2, but not Slc11a1, can complement EGTA sensitivity in smf1Delta/smf2Delta/smf3Delta yeast. However, some studies of transport in mammalian cells suggest Slc11a1 is a symporter. We now demonstrate that Slc11a1, but not Slc11a2, complements a divalent cation stress phenotype in bsd2Delta/rer1Delta yeast. This is the first description of a yeast complementation assay for Slc11a1 function. Given the prior demonstration in frog oocytes that Slc11a1 acts as an antiporter, the most plausible interpretation of the data is that Slc11a1 is rescuing bsd2Delta/rer1Delta yeast by exporting divalent cations. Chimaeras define the N terminus, and a segment of the protein core preceding transmembrane domain 9 through transmembrane domain 12, as important in rescuing the divalent cation stress phenotype. EGTA sensitivity and divalent cation stress phenotypes in yeast expressing Slc11a orthologues show that symport activity is ancestral. Molecular changes that mediate rescue of the divalent cation stress phenotype post-date frogs and co-evolved with Slc11a1 orthologues that regulate divalent cation homeostasis in macrophages and resistance to infection in chickens and mammals.
Highlights
These results are analogous to our demonstration [18] that murine Slc11a1 and human SLC11A1 expressed in frog oocytes flux divalent cations in either direction across the oocyte membrane against a proton gradient
At raised extracellular pH there was a net influx of divalent cations into frog oocytes; at low extracellular pH there was a net efflux of divalent cations from frog oocytes
These observations led us to conclude that murine and human Slc11a1/SLC11A1 molecules behaved as proton/divalent cation antiporters. Given these prior observations in frog oocytes, and the demonstration here using the zinc responsive elements (ZREs)-lacZ reporter construct that Slc11a1 expressing yeast cells had lower intracellular Zn2ϩ than non-transformed cells and cells expressing Slc11a2, the most plausible interpretation of our yeast data is that Slc11a1 is acting as an antiporter to transport divalent metal cations out of the yeast cell under heavy metal stress conditions
Summary
This provides an explanation for data showing that murine/human Slc11a2/SLC11A2, but not Slc11a1/ SLC11A1, can complement EGTA and pH sensitivity phenotypes in yeast deleted for the Slc11a homologues SMF1/2/3, even though both were equivalently expressed at the yeast plasma membrane [19, 20] It follows from the frog oocyte studies that Slc11a2 would transport divalent cations with the proton gradient from acidic intracellular vesicles of mammalian cells to the cytoplasm. If our previous observation [18] of antiport function in frog oocytes was correct, it should be possible to develop such an assay under conditions of divalent cation stress, as opposed to the Mn2ϩ depletion and EGTA sensitivity generated by knock-out of the yeast Slc11a orthologues SMF1/2/3 [19, 20] To accomplish this we took advantage of the observation that, under metal replete conditions, the Bsd protein regulates metal homeostasis in yeast by trafficking Smf1p and Smf2p to the vacuole for degradation [25]. Functional phylogenetic analysis shows that symport activity is ancestral, with ability to rescue a divalent cation stress phenotype coevolving with Slc11a1 orthologues in chickens, mice, and humans
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