Figure 4. A conformational switch underlies Mup1 function and turnover.

• A, B
  Comparisons of the functional activities of different Mup1 mutants based on direct quantification of ¹⁴C‐Met uptake (A) and growth sensitivity to the toxic Met analog selenomethionine (SeMet, B). Growth assay is shown from top to bottom as a fivefold dilution series. White separator line indicates border to a lane that was removed from the original plate image.
• C, D
  Endocytic internalization of the different Mup1 mutants fused to GFP. Ratios of PM to cytosolic fluorescence intensities (C) and representative images at equatorial planes (D) are shown.
• D
  Western blot showing ubiquitination (Ub) of the indicated Mup1 mutants grown in the absence (−) and presence of Met (+, 5 min after addition). Loading control as in Fig 1C. Mutants utilized: wt, wild‐type Mup1; ΔC, deletion of the C‐terminal segment to aa519; W155A and G78N, point mutants; ΔN, deletion of the N‐terminal segment to aa52; FWRV, mutation of aa534–537 to alanines; 2KR, Mup1 with N‐terminal mutations K27R and K28R. All strains refer to Mup1 variants fused to C‐terminal GFP.

Data information: Values are means ± SD, n = 2–4 experiments (A) and n = 50–250 cells (C). n.d.: not determined. Scale bars: 2 μm. All measured values are listed in Table EV1.Source data are available online for this figure.