Mutational analysis of Saccharomyces cerevisiae Smf1p, a member of the Nramp family of metal transporters.
We have recently shown that a member of the Nramp family of metal transporters, Saccharomyces cerevisiae Smf1p, is tightly regulated at the level of protein stability and protein sorting. Under metal replete conditions, Smf1p is targeted to the vacuole for degradation in a manner dependent on the S. cerevisiaeBSD2 gene product, but under metal starvation conditions, Smf1p accumulates at the cell surface. Here, we have addressed whether Smf1p activity may be necessary for its regulation by metal ions and Bsd2p. Well conserved residues within transmembrane domain 4 and the transport signature sequence of Smf1p were mutagenized. We identified two mutants, G190A and G424A, which destroyed Smf1p activity as monitored by complementation of a smf1 mutation. Notably, these mutations also abolished control by metal ions and Bsd2p, suggesting that Smf1p metal transport function may be necessary for its regulation. Two additional mutants isolated (Q419A and E423A) exhibited wild-type complementation activity and were properly targeted for vacuolar degradation in a Bsd2-dependent manner. However, these mutants failed to re-distribute to the plasma membrane under conditions of metal starvation. A model is proposed herein describing the probable role of Smf1 protein conformation in directing its movement to the vacuole versus cell surface in response to changes in metal ion availability.
Liu
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XF
Department of Environmental Health Sciences, Johns Hopkins University School of Public Health, Baltimore, MD, 21205, USA.
Culotta
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VC
D000483:Alleles; D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D027682:Cation Transport Proteins; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D017136:Ion Transport; D033862:Iron-Binding Proteins; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D008670:Metals / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D000099173:Natural Resistance-associated Macrophage Protein 1; D000099236:Solute Carrier Family 11, Member 2
D002352:Carrier Proteins; D027682:Cation Transport Proteins; D005656:Fungal Proteins; D033862:Iron-Binding Proteins; D008565:Membrane Proteins; D008670:Metals; D029701:Saccharomyces cerevisiae Proteins; D000099173:Natural Resistance-associated Macrophage Protein 1; D000099236:Solute Carrier Family 11, Member 2; C105116:SMF1 protein, S cerevisiae