Disparate roles of the Nramp transporters, Smf1p and Smf2p. Smf1p (in red), which has been found to localize both at the cell surface and in intracellular vesicles (depicted as circles), is primarily responsible for providing manganese to a nonproteina-ceous manganese-requiring anti-oxidant that can substitute for Cu/Zn SOD1.15 Smf2p (in green), which is found primarily in intracellular vesicles, but is also thought to reside at the cell surface in minute quantities, is responsible for total cellular manganese accumulation. The manganese transported by Smf2p is bioavailable to manganese-requiring enzymes such as sugar transferases (STase) and Sod2p, which are present in the Golgi and mitochondria, respectively.

Multiple tiers of regulation of the Smf proteins. (A) When manganese is sufficiently available to cells, under nontoxic concentrations, ~90% of Smf1p (red), is targeted to the vacuole immediately after synthesis by Rsp5p (purple) mediated ubiquiti-nation. This ubiquitination is facilitated by the Trep (blue)–Bsd2p (gold) adapter complex. However, the remaining Smf1p is sorted to either the cell surface or intracellular vesicles.13,19,39–41,56 (B) When cells are starved for manganese, Smf1p (red) is not sent for degradation to the vacuole but is instead sorted to the cell surface.13,19,31,39 (C) When cells are exposed to excess toxic levels of manganese, the degradation of Smf1p (red) is biphasic. In the fast step, cell-surface Smf1p is subject to rapid endocytosis and movement to the vacuole in a Rsp5p-dependent manner. In the slow step, intracellular Smf1p undergoes a relatively slow Rsp5p-independent movement toward the vacuole that only occurs with prolonged exposures to toxic manganese.33 (D) When cells are exposed to toxic cadmium, but not manganese, cell surface Smf1p (red) is endocytosed to the vacuole for degradation.59 This occurs in a Trep–Bsd2p independent fashion, instead requiring Ecm21p or Csr2p (green) as adapters for Rsp5p (purple).

Comparison of manganese transport and trafficking between S. cerevisiae (left) and metazoans (right). In yeast, Smf2p (green) provides the bulk of total cellular manganese (pink balls), which is bioavailable to manganese requiring enzymes. Smf1p (red) provides manganese to the manganese anti-oxidant and assists Smf2p import of manganese when cells are manganese starved. Pho84p (gold), the manganese phosphate transporter, is responsible for importing manganese under conditions of high extracellular toxic concentrations of manganese. The vacuolar manganese transporter, Ccc1p (gold rectangle), and the newly identified putative vacuolar manganese importer, Ypk9p (maroon rectangle), help to detoxify toxic manganese. Likewise, manganese pumped into the Golgi via Pmr1p (purple) is detoxified by being exocytosed out of the cell. In metazoans, the Nramp homologue Dmt1 (red), which is expressed in the duodenum, is responsible for dietary iron and manganese intake. The other Nramp homologue, Nramp1 (green), which is expressed in the phagosome of macrophages, serves to pump out iron and manganese that invading microbes require to survive. The basal ganglion is especially sensitive to manganese toxicity due to the expression of Dmt1 in the brain. Like yeast, toxic manganese in metazoans may be detoxified by vacuolar sequestration via the Ypk9p homologue, ATP13A2 (maroon rectangle), or excreted out of the cell by the Pmr1p homologue, SPCA2 (purple).

Approximation of the intracellular accumulation of manganese in yeast as a function of varying concentrations of manganese supplemented to a minimal medium. Superimposed on the diagram is a growth isotherm of yeast cells as a function of manganese supplementation. The red bars on the growth isotherm correspond to metal-induced toxicity due to manganese deficiency or surplus, while the green bar corresponds to growth under conditions of sufficient, i.e., nontoxic, manganese.

Bsd2p regulation of the Smf proteins. When manganese is sufficiently available to cells, the Smf proteins (combination of Smf1p and Smf2p, in red) in the active metal bound state are ubiquitinated (Ub, yellow) by Rsp5p (purple) in a Bsd2p (gold) and Trep1 and Trep2 (collectively shown here as Trep; blue) dependent manner and directed to the vacuole for degradation. First, Rsp5p and Bsd2p interact through their PY domains, forming a complex. This complex next recognizes the PY domain of Trep via a recognition site on Rsp5p. The Trep/Bsd2p/Rsp5 complex then mediates the ubiquitination of the manganese-bound Smf proteins, leadingtotheirtargetingtothevacuolefordegradation.39–41,56

Proposed model for the roles of Pmr1p and Smf1p in providing manganese to the manganese anti-oxidant.15 Manganese that is provided by Pmr1p (purple) to the Golgi is recycled by Smf1p (red) for its use by the manganese anti-oxidant.