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 transport ers, Saccharomyces cerevisiae Smf1p, is tightly regulated at the level of p rotein stability and protein sorting. Under metal replete conditions, Smf1p is targeted to the vacuole for degradation in a manner dependent on the S. cerevisiae BSD2 gene product, but under metal starvation conditions, Smf1p accumulates at the cell surface. Here, we have addressed whether Smf1p act ivity may be necessary for its regulation by metal ions and Bsd2p. Well con served residues within transmembrane domain 4 and the transport signature s equence of Smf1p were mutagenized. We identified two mutants, G190A and G42 4A, which destroyed Smf1p activity as monitored by complementation of a smf 1 mutation. Notably, these mutations also abolished control by metal ions a nd Bsd2p, suggesting that Smf1p metal transport function may be necessary f or its regulation. Two additional mutants isolated (Q419A and E423A) exhibi ted wild-type complementation activity and were properly targeted for vacuo lar degradation in a Bsd2-dependent manner. However, these mutants failed t o re-distribute to the plasma membrane under conditions of metal starvation . A model is proposed herein describing the probable role of Smf1 protein c onformation in directing its movement to the vacuole versus cell surface in response to changes in metal ion availability. (C) 1999 Academic Press.