FIG. 4.

MT binding of KLC3. A) In vitro-translated, radiolabeled KLC3 was incubated with purified, taxol-stabilized MTs and pelleted. The MT pellets were washed and repelleted twice more, after which the pellet and the three supernatants were examined for tubulin (upper panel, coomassie-stained region of the gel) and KLC3 (lower panel, autoradiogram). Lane 1: aliquots of reaction mixture of translation mix and MT (mix); lanes 2–4: aliquots of supernatants from the first, second, and third pelleting reaction (sup1–sup3); lane 5: final pellet (pellet). Tubulin and KLC3 are indicated. B) To analyze ATP-dependency of the KLC3-MT association, the above protocol was repeated in the presence of either AMP-PNP or indicated amounts of ATP. Aliquots of the supernatant and the entire pellet were analyzed by SDS-PAGE. KLC3-containing gel slices were identified by autoradiography, excised from the gel, and counted. The amount recovered is shown. Supplanting AMP-PNP with ATP resulted in a shift of KLC3 from the MT pellet to the supernatant.