Figure 5. ZIP13ΔFLA protein is degraded by a proteasome-dependent pathway.
A Location of the ΔFLA mutation (deletion of phenylalanine–leucine–alanine in TM3) in ZIP13.
B Amino acid alignment of the TM3 of human ZIP family members. Amino acids conserved in all of the indicated zinc transporters (*), conserved substitutions (:), semi-conserved substitutions (.). Red: hydrophobic amino acids; blue: acidic amino acids; magenta: basic amino acids; green: hydrophilic amino acids.
C Protein expression level of G64D-V5 in 293T stable lines. The cell lysates of two representative clones stably expressing WT-V5 or the G64D-V5 mutant were analyzed by Western blot using an anti-V5 antibody.
D Protein expression level of ΔFLA-V5 in 293T stable lines. The cell lysates of two representative clones stably expressing WT-V5 or the ΔFLA-V5 mutant were analyzed by Western blot using an anti-V5 antibody.
E The hCD8 expression levels in 293T stable lines, as an indicator of the amount of transfected plasmid DNA (pMX-WT-IRES-hCD8, pMX-G64D-IRES-hCD8, or pMX-ΔFLA-IRES-hCD8). Two representative clones stably expressing WT-V5 or the G64D-V5 or ΔFLA-V5 mutant were analyzed by flow cytometry using an APC-conjugated anti-hCD8 antibody. Histograms were gated on hCD8-positive cells.
F Recovery of mutant ZIP13 protein expression by MG132 treatment. Representative 293T clones stably expressing WT-V5 (#1), G64D-V5 (#1), or ΔFLA-V5 (#2), were treated with 10 μM MG132 for the indicated times, followed by Western blotting analysis with an anti-V5 antibody.
G Posttranslational degradation of mutant ZIP13 proteins. HeLa clones stably expressing WT-V5, G64D-V5, or ΔFLA-V5 were treated with 10 μM CHX for the indicated times. Total cell lysates were analyzed by Western blot using an anti-V5 antibody (upper). Right graph shows the relative expression level of ZIP13 proteins over time. Data are representative of three independent experiments.
H Protein expression level of the SCD-EDS pathogenic mutants in human fibroblasts in the presence of bortezomib. Human dermal fibroblasts transiently expressing ZIP13 mutants were treated with 10 nM bortezomib for 6 h, followed by Western blotting analysis using an anti-V5 antibody.
Source data are available online for this figure.