Abstract. Axoplasmic vesicles were purified and observed to translocate on isolated microtubules in an ATP-dependent, trypsin-sensitive manner, implying that ATP-binding polypeptides essential for force generation were present on the vesicle surface. To identify these proteins [a32p]8-azidoadenosine 5'-triphosphate ([ct32p]8-N3ATP), a photoaflinity analogue of ATE was used. The results presented here identify and characterize a vesicle-associated polypeptide having a relative molecular mass of 292 kD that bound [a32p]8-N3ATP. The incorporation of label is ultraviolet light-dependent and ATP-sensitive. Moreover, the 292-kD polypeptide could be isolated in association with vesicles or microtubules, depending on the conditions used, and the data indicate that the 292-kD polypeptide is similar to mammalian brain microtubuleassociated protein 2 (MAP 2) for the following reasons: The 292-kD polypeptide isolated from either squid axoplasm or optic lobe cross-reacts with antiserum to porcine brain MAP 2. Furthermore, it purifies with taxol-stabilized microtubules and is released with salt. Based on these characteristics, the 292-kD polypeptide is distinct from the known force-generating molecules myosin and flagellar dynein, as well as the ll0-B0-kD kinesin-like polypeptides that have recently been described (Brady, S. T., 1985, Nature (Lond.), . Because the 292-kD polypeptide binds ATP and is associated with vesicles that translocate on purified MAP-free microtubules in an ATP-dependent fashion, it is therefore believed to be involved in vesicle-microtubule interactions that promote organdie motility.
DRECTED intracellular organelle movement is a fundamental process characteristic of all cells. This process has been well studied in the neuron because membranous organelles must travel very long distances, up to a meter or more in length, between the cell body and the synaptic terminal. This process, called fast axonal transport, is ATP-dependent, rapid (1-5 lxm/s), and selective. Newly synthesized membranous organelles produced in the cell body are transported in the orthograde direction toward the nerve terminal. The organelles that are moving in the retrograde direction, however, are different in nature and are being transported from the synaptic terminal region back to the cell body for degradation (reviewed in Grafstein and Forman, 1980;Weiss, 1982). The giant axon of the squid Loligo pealei has been an important preparation for the study of the molecular basis of such organelle transport. The axon is unmyelinated, up to 1 mm in diameter, and 4-8 lxl of axoplasm can be mechanically extruded away from the surrounding plasma membrane and glial sheath (Bear et al., 1937;Lasek, 1974). Moreover, the preparation remains metabolically active for hours, accessible to surrounding solutions for experimentation, and the organelle motility can be observed directly using video-enhanced differential interference contrast microscopy . These characteristics of the axoplasm from Loligo pealei have enhanced the analysis of the relati...