Figure 3

Cleavage of the presequence occurs around residue 32 in the N-terminal region of NDUFV2. (a) Two possible mitochondrial processing sites of NDUFV2 were predicted by the TargetP server [36]. The diagram shows a part of the N-terminal sequence of NDUFV2 (residues 17-51), with the MPP and MIP consensus cleavage sequence, R-10 motif (xRx↓(F/L/I)xx(S/T/G)xxxx↓), above it. The arrows indicate the expected MPP and MIP cleavage sites on NDUFV2. (b) The cleavage site of NDUFV2 in vivo is located around amino acid residue 32. Lanes 1-5, the total cell lysates of T-REx-293 transfected with the c-myc-tagged full-length NDUFV2 (lanes 1 and 5) and the c-myc -tagged NDUFV2 lacking the first 18, 32, and 50 residues respectively (lanes 2-4). Cell lysates were resolved by 15% SDS-PAGE, transferred, and probed with a mouse monoclonal anti-c-myc antibody. β-actin (42 kDa) was used as an internal control for Western blotting. (c) Mutation of the -10 arginine alone (i.e. R23A mutation) in the precursor has little effect on the formation of mature NDUFV2. Western blot analyses were conducted using mitochondrial extracts from T-REx-293 cells transiently transfected with the wild-type (lane 1) or NDUFV2 R23A mutant (lane 2) construct. The expressed proteins were detected by an anti-c-myc antibody. ATP synthase subunit α (ATP α) was used as a mitochondrial marker.