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. 2017 Oct 5;6:e29985. doi: 10.7554/eLife.29985

Figure 4. Conservation of ATP and substrate binding mechanism in hPINK1.

(A) hPINK1 knock out HeLa cells transiently co-expressing WT human Parkin and 3xFLAG tagged hPINK1 WT, KI or hPINK1 ATP-binding mutants A217D, K219A, E240K, L369P. A217, K219, E240 and L369 correspond to A194, K196, E217 and L344 of TcPINK1, respectively. Cells were stimulated with 10 μM CCCP or DMSO for 3 hr. Lysates were subjected to immunoblotting as follows: pSer65 Parkin (anti-phospho-Parkin); Parkin (anti-Parkin), pSer65 ubiquitin (anti-phospho-ubiquitin), hPINK1 (anti-FLAG), OPA1 (anti-OPA1) and GAPDH (anti-GAPDH). (B) hPINK1 knock out HeLa cells transiently co-expressing WT human Parkin and 3xFLAG tagged hPINK1 WT, KI or hPINK1 Ins mutants ∆Ins1 (180–209 deletion), ∆Ins2 (245–265 deletion), ∆Ins3 (285-294). Cells were stimulated with 10 μM CCCP or DMSO for 3 hr. Lysates were subjected to immunoblotting as follows: pSer65 Parkin (anti-phospho-Parkin); Parkin (anti-Parkin), pSer65 ubiquitin (anti-phospho-ubiquitin), hPINK1 (anti-FLAG), OPA1 (anti-OPA1) and GAPDH (anti-GAPDH).

Figure 4.

Figure 4—figure supplement 1. Sequence alignment of TcPINK1 and hPINK1.

Figure 4—figure supplement 1.

Secondary structure elements are included based on TcPINK1 structure. Elements numbering is consistent with Figure 1C; numbering of residues is according to the TcPINK1 sequence. ATP-binding residues shown in Figure 1 are marked with *. Locations of loop insertions are coloured according to Figure 1.