Downregulation of Smf1 requires Bsd2 and a Tre protein. (A) Diagram of Tre1 and Tre2 compared to human TFR. The Y-x-x-hydrophobic motif (Y), PPxY motif, PA domain, M28 protease domain and TFRD are indicated. (B) Sensitivity to cadmium. Cells of the indicated strains were grown on plates containing 20 μM cadmium chloride. Vertical columns represent serial dilutions of cells. All strains were plated at equivalent densities. (C) Complementation by Tre proteins and their GFP-tagged derivatives; assay as in panel B. (D) Immunoblot of GFP-Smf1 expressed in the indicated strains. Phosphoglycerate kinase (Pgk1) was detected on the same blot as a loading control. Cells were grown in YEPD medium, which is metal-replete. (E) GFP-Smf1 imaged in the indicated strains. Arrows indicate plasma membrane fluorescence, which was variably seen in both tre1 tre2 and bsd2 cells.

Tre proteins can affect Smf activity in the absence of Bsd2. (A) Overexpression of Bsd2 cannot restore cadmium resistance of a tre1 tre2 strain. Bsd2 was expressed from the TPI1 promoter, which results in at least 10-fold overexpression (), in the indicated strains. (B) Tre proteins, expressed from the TPI1 promoter, can suppress cadmium sensitivity of a bsd2 mutant. (C) Tre1 overexpression does not reduce Smf1 levels in a bsd2 mutant. Tre1 (left) and mutant versions of it (right, PPAG and Δ52 Δlum) were expressed from the TPI1 promoter in cells of the indicated strains that were also expressing Smf1-HA. Bsd2 was similarly expressed. The Smf1 was detected by alkaline lysis of the cells and immunoblotting with anti-HA. The same blot was probed with a rabbit antibody that recognises an unknown minor yeast protein, as loading control. No HA refers to a control strain that lacked the tagged Smf1. The right-hand panels and the left-hand panel are from two different gels. (D) Overexpression of Tre1 reduces uptake of Mn by Bsd2 mutant cells. Cells of the indicated strains were grown in metal-replete medium and ⁵⁴Mn uptake measured. The results are the mean of two separate experiments, each in duplicate. Bars indicate standard error of the mean.

Binding and ubiquitination. (A) The PPxY motif binds the Rsp5 WW domains. Protein A fusions of the entire cytoplasmic domain of Tre1, with the wild-type PPVY sequence or a mutated PPAG motif, were incubated with GST fusions of each of the three WW domains of Rsp5 and bound protein detected by immunoblotting. (B) Tre1 is ubiquitinated. Left panel: a GFP-tagged version of Tre1 that lacked the luminal domain was detected either by immunoblotting of cell lysates with anti-GFP or by immunoblotting with anti-ubiquitin after immunoprecipitation with anti-GFP. The positions of size markers of 53 and 93 kDa are indicated. Right panel: full-length wild-type Tre1 (PPVY) and a PPxY mutant (PPAG), tagged with protein A, were expressed in the indicated strains and detected by immunoblotting. Ubiquitinated protein is indicated by Ub. The anti-ubiquitin antibodies do not efficiently recognise protein with a single ubiquitin moiety. (C) Tre1 binds Bsd2 in vivo. Cells expressing HA-tagged Bsd2 and either endogenous Tre1 only (Control) or a protein A-tagged Tre1 construct lacking the luminal domain were lysed in detergent (total), protein A-tagged Tre1 purified on IgG Sepharose beads and bound HA-Bsd2 detected by immunoblotting. (D) Smf1-HA was expressed, immunoprecipitated with anti-HA and immunoblotted. The left panel shows protein from BSD⁺ (+) and bsd2 cells. The right panel shows material from cells expressing myc-ubiquitin, probed simultaneously with anti-HA and anti-myc; the two fluorescent channels are shown side by side. Ub indicates ubiquitinated forms and the asterisk indicates polyubiquitinated protein. All yeast strains used for this figure were pep4.

Tre1 is sorted to the vacuole by Bsd2. (A, B) GFP-Tre1, or the PPAG mutant, expressed from the TPI1 promoter in the indicated strains. (C) Double label of GFP-Tre1 in bsd2 cells that had been incubated for 10 min with FM4-64. (D) Immunoblot of GFP-Tre1, detected with anti-GFP, in the indicated strains. The left-hand panel shows proteins expressed from the TPI1 promoter and the right-hand panel shows GFP-Tre2 expressed from its own promoter, exposed for much longer, as the signal was very weak. Bars in A and C, 3 μm.

Tre1 mutants. (A) Diagrams of the various Tre1 mutants tested. The luminal domain is not shown. The regions implicated in various processes, as inferred from tests of these mutants, are summarised at the bottom. (B) Sequences of the cytoplasmic domains of Tre1 and Tre2 showing identities and deletion end points. Lines correspond to the features indicated by black boxes in panel A.

Sorting of Tre1 mutants. (A) Effect of the TMD on sorting of truncated Tre1 to the vacuole in wild-type cells. GFP-Tre1 constructs that lacked the luminal domain (Δlumen) or in addition had the TMD replaced by that of Pep12 or Sso1 are shown. Bar, 3 μm. (B) GFP-tagged cytoplasmic domain mutants of Tre1 expressed in wild-type (WT) bsd2, tul1, and bsd2 tul1 double mutant cells. See for details of the mutations. (C) Immunoblot of the mutants expressed in tre1 tre2 cells. The prominent band at 33 kDa is free GFP generated by vacuolar proteolysis.

Regions of Tre1 required for Smf1 regulation. (A) Suppression of bsd2 cadmium sensitivity by overexpressed protein. GFP-tagged Tre1 and derivatives of it (see for details) were expressed from the TPI1 promoter in the indicated strains. Growth was on plates containing 20 μM cadmium. (B) Suppression of tre1 tre2 cadmium sensitivity by Tre1 derivatives expressed from the TPI1 promoter. (C) Suppression of tre1 tre2 cadmium sensitivity by Tre1 mutants expressed from the TRE1 promoter, at low levels.

Model for Tre protein function and relationship to the TFR. (A) Model. Smf1, in a metal-bound conformation, is recognised by Tre1. Tre1 is in a complex with Bsd2, which, through the PPxY motifs of both proteins, recruits Rsp5. Rsp5 ubiquitinates all three membrane proteins, which are thereby directed into the MVB pathway to the vacuole. (B) Evolutionary relationships of the Tre proteins. A family tree of ascomycete proteins that share the domain structure of the Tre proteins, together with human relatives, was prepared with the program clustal W using related proteins from Physarum and Gloeobacter as outliers. Proteins were categorised according to the presence of six residues shown to be crucial for protease activity. Solid lines indicate proteins likely to be active proteases. Dotted lines indicate a non-functional protease domain, and dashed lines indicate both lack of protease activity and the presence of a PPxY motif in the cytoplasmic domain. A, Aspergillus; C, Candida; D, Debaromyces; E, Eremothecium; G, Gibberella; H, Homo; K, Kluyveromyces; M, Magnaporthe; N, Neurospora; S, Schizosaccharomyces (pombe only) or Saccharomyces; Y, Yarrowia.