Figure 6
Late domains of CLIC4 modulate the LE lumen sorting step of MMP14 trafficking. (A) Amino acid alignments of CLIC4 from different species. The two overlapping late domains (PPXY and YXXL) are highly conserved in vertebrates, but less conserved in invertebrates. (B) The putative dual late domain sequence in human CLIC4. The CLIC4 protein structure was imaged (Protein Data Bank ID: 2AHE. Doi: 10.2100/pdb2ahe/pdb) using online JSmol software. (C) (Left panels) The representative immunoblots of total lysates of 293 T cells transfected with mCherry-Tsg101 together with Flag-CLIC4-WT or Flag-CLIC4-Y104A and probed by the indicated antibodies. (Middle panel) The immunoblots of the immunoprecipitants pulled down by the control or anti-Flag antibody from the indicated transfected cell lysates. (Right panel) Immunoblots of Flag immunoprecipitants isolated from the indicated transfected cell lysates. Compared to its WT counterpart, the Flag-CLIC4-Y104A pulled down less mCherry-Tsg101. (D) The total lysates (left panels) as well as the immunoprecipitants pulled down by anti-Flag or control antibody (right panels) were immunoblotted by the indicated antibodies. Asterisks point to the MMP14-mCherry species that was specifically co-immunoprecipitated with Flag-CLIC4. (E) ARPE19 cell line expressing inducible CLIC4-sh was treated with Dox for 3 days and then transfected with MMP14-mCherry, GFP-CD63, together with CLIC4-WT-IRES-FLAG-Rab5 Q79L, or CLIC4-Y104A-IRES-FLAG-Rab5 for 1 day in the same Dox conditions. Representative confocal images of mCherry and GFP stained cells are shown. Arrowheads and arrows point to LE luminal and LE limiting membrane signals of MMP14, respectively. (F) MMP14 intensity ratio in LE lumen for (E). n = 120 endosomes from 24 cells. Bars show means ± S.D. from three independent experiments. p value, t-test. Scale bar = 10 µm. (G) Immunoblots demonstrate the same amount of ectopically expressed proteins in the rescue experiments (in E,F).
