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. 2010 Nov 29;191(5):933–942. doi: 10.1083/jcb.201008084

Figure 3.

Figure 3.

Point mutations in the transmembrane domain of PINK1 partially inhibit its proteolytic cleavage. (a) Amino acids throughout the predicted transmembrane domain of PINK1 were mutated to phenylalanine (amino acids 91–98) or tryptophan (amino acids 99–110). (b) HeLa cells transfected with the indicated PINK1-YFP mutants were treated with either DMSO, 10 µM CCCP, or 10 µM MG132 for 3 h. 20 µg cell lysates were subjected to SDS-PAGE and immunoblotting using antibodies against PINK1 and tubulin. (c) The band intensity of FL PINK1 in DMSO- or CCCP-treated lanes from b was densitometrically measured using Multi Gauge (Fujifilm). After corrections for background and loading, the band intensity ratio of DMSO/CCCP-treated samples for each PINK1 mutant was measured. (d) YFP-tagged WT PINK1 and PINK1 R98F mutants were transfected into HeLa cells. Cells were stained with MitoTracker red before treatment with 10 µM CCCP for 3 h and analyzed by confocal microscopy. (bottom) Enlarged views of the boxed areas are shown. Bars, 20 µm. Green arrowheads, FL and ΔMTS-PINK1; red arrowheads, 52-kD PINK1.