ADCK3 contains an N-terminal MTS.

(A, B). An N-terminal FLAG tag does not prevent entry of ADCK3 into mitochondria. HeLa cells transiently transfected with pcDNA3.1/Hygro(+) only (Vec), pcDNA3.1/Hygro(+)-FLAG-ADCK3 (FLAG-ADCK3) or pcDNA3.1/Hygro(+)-ADCK3-FLAG (ADCK3-FLAG). Immunofluorescence (A) or immunoblotting of WCEs (B) was performed with antibodies to ADCK3, FLAG and β actin. ADCK3* panel is a longer time exposure. Counterstaining for mitochondria was performed with Mitotracker Deep Red (Mitotracker). Nuclei were stained with Hoechst 3342. White bars: 15 μm. Note: The absence of peri-nuclear staining in Vec transfected cells was due to the reduced exposure time needed to prevent saturation of fluorescence from FLAG-ADCK3 and ADCK3-FLAG transfected cells. (C). ADCK3 contains two predicted alpha helical domains at its N-terminus. ADCK3 primary sequence (only αα1–350 are shown) was inserted into the online secondary structure prediction tools PROF and PNSS (ProteinPredict server). Blue boxes: predicted alpha helical regions. Orange box: Region of non-regular secondary structure. Dashed green box: Motif specifically related to CoQ biosynthesis. Dashed blue box: kinase-like domain. Arrow indicates the position of a possible MPP site. (D). A helical wheel arrangement of the N-terminal region of ADCK3 shows slight amphipathicity. The amino acids from the first predicted alpha helix described in C were inserted into an online tool to produce the helical wheel arrangement of residues depicted (see Experimental Procedures). Blue Squares: nonpolar residues. Red Diamonds: acidic residues. Black circles: basic residues. (E). ADCK3 undergoes a single cleavage event on entry into mitochondria. In vitro import of S35-methionine labelled ADCK3 into isolated mitochondria. 10 μM CCCP used to ablate ΔΨm. Lysate lane shows separation of in vitro translated protein only. Autorad image is depicted. p: precursor. m: mature protein. PK: proteinase K. (F). N-terminal sequencing of mature ADCK3. Schematic of ADCK3 indicating the position of the cleavage event observed in B as determined by N-terminal sequencing. R residues observed at positions -2, -3 and -13 are consistent with the known features of an MPP cleavage site. (G). Alignment of cleavage sequence positions amongst ADCK3 orthologues.