SNPH inhibits KIF5 motor ATPase activity. (A and B) Deleting snph has no effect on the motor association with axonal mitochondria. Axonal mitochondria were isolated from sciatic nerves of snph^+/+ and snph^−/− mice. 15 µg of total proteins from supernatant and mitochondria-enriched fractions was loaded in SDS-PAGE and sequentially immunoblotted on the same membrane after stripping between each antibody application. The intensities of KIF5 and dynein IC74 were normalized to mitochondrial marker Tom20. Relative purity of the mitochondrial fraction was assessed by sequential immunoblotting mitochondrial marker Tom20, SNPH, and other markers including EEA1 (endosomes), Grp78 (ER), and actin. Data were collected from four paired littermates. Error bars show SEM; Mann–Whitney U test. (C) SNPH and Trak2 play opposite roles in regulating KIF5C ATPase. ATPase activity of full-length KIF5C or its motor domain (KIF5C–N-MD) was measured using Kinesin ATPase Endpoint Biochem kit and expressed as hydrolyzed ATP. MT-stimulated ATPase activity of both KIF5C (blue) and KIF5C–N-MD (green) was used as a reference value for subsequent endpoint assays by adding SNPH (or its mutant) or Trak2 under the same conditions. Incubating SNPH (1–469 aa) significantly reduces the motor ATPase activity (P = 0.029), whereas Trak2 increases the activity (P = 0.028). Deleting the KIF5 binding domain (ΔKBD) abolishes its inhibitory role. Error bars show SEM. Mann–Whitney U test, n = 4. (D) Representative time-lapse imaging showing the movement of EGFP-KIF5C along MTs labeled with mCherry–α-tubulin in a live COS cell. (E) Mean velocity of EGFP-KIF5C puncta along MTs in living cells with or without expression of SNPH, its ΔKBD mutant, or Trak2. Note that coexpressing SNPH immobilizes KIF5C puncta. Data were quantified from a total number of EGFP-KIF5C puncta (indicated in parentheses above the bars). Error bars show SEM. Student’s t test. WT, wild type; KO, knockout.