Fig. 4. Binding of microtubules to mitochondria in vitro. (A) Isolated mitochondria were incubated with and without an excess of microtubules and subjected to floatation centrifugation in a sucrose density gradient. After harvesting fractions of the gradient were precipitated with TCA and analysed by SDS-PAGE and Coomassie staining. Tubulin floated with mitochondria is marked with an asterisk. (B) Isolated mitochondria were incubated with microtubules, and floated in a sucrose density gradient; fractions were harvested, proteins were precipitated and analysed by SDS-PAGE and western blot. Only fractions 1 (containing floated mitochondria as controlled by blotting against porin), 3 (from the middle of the gradient) and 5 (containing non-floated proteins) are shown. P, pellet from the bottom of the gradient. Top panel, standard: mitochondria incubated with microtubules under standard conditions (i.e. in the absence of adenine nucleotides and without pretreatment of organelles). Panels 2 and 3: trypsin-pretreated mitochondria and salt-washed mitochondria, respectively, which were incubated with microtubules under standard conditions. Panels 4 and 5: non-pretreated mitochondria that were incubated with microtubules in the presence of ATP or ADP, respectively.

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