Kinesin is a microtubule-based motor protein that transports organelles to the plus ends of microtubules. Kinesin was first identified in neural tissue as the protein responsible for anterograde fast axonal transport, the movement of membrane bounded organelles toward the synapse in neurons. The molecule has a native molecular weight of ~360kDa and is a heterotetramer composed of two identical heavy chains of ~120kDa and two accessory 64-kDa light chains. The heavy chain has an amino-terminal globular domain of ~40kDa, a central region that forms coiled coils, and a C-terminal region. The central regions of two heavy chains dimerize, and the kinesin dimer is an ~100nm rod that has two globular domains at one end (the heads) and a fan-shaped tail at the other (Fig. 1). The light chains are located at the tail. The head domains generate the motive force, bind microtubules, and hydrolyze ATP. The heavy chain tail and the light chains are important for binding kinesin to cargo. In the presence of the nonhydrolyzable ATP analog AMPPNP (5′ adenylylimidodiphosphate), kinesin binds tightly to microtubules. This property is used to purify kinesin from other microtubule-binding proteins. When kinesin is attached to a glass coverslip to which microtubules and ATP are added, microtubules can be observed moving along the surface of the coverslip. In this in vitro microtubule gliding assay, kinesin moves microtubules at a velocity of ~0.5microns/second.
Figure 1. Schematic illustration of the overall structure of a kinesin.
Immunofluorescence microscopy with antibodies to the heavy and light chains localizes kinesin to membranous organelles in cultured cells. Antibodies to the kinesin heavy chain inhibit organelle movement in vivo (1), and antisense probes to the kinesin heavy chain gene block anterograde axonal transport in cultured neurons (2). Mutations of the kinesin heavy chain also disrupt axonal transport (3).
Kinesin was the first member of the rapidly growing kinesin superfamily identified. The different members of the superfamily are sometimes called Kifs (kinesin family members). Each family member shares the conserved heavy chain motor (head) domain, which binds microtubules and hydrolyzes ATP. However, the family members differ in the structure of the rest of the heavy chain and the composition of the accessory proteins. The location of the motor head domain is not conserved. The head domain of different family members can be found at either the amino terminus, the carboxy terminus, or in the center of the polypeptide chain. The archetypal kinesin is involved only in movement of membrane-bounded organelles, but many of the other kinesin family members are involved in assembling and maintaining the spindle and in chromosomal movement. Several of the kinesin family members have been well characterized, and a description of three members demonstrates the variety of molecules and their functions. The BimC family of kinesins is important for chromosomal separation in mitosis and in maintaining spindles. They are composed of four ~120kDa heavy chains that form a bipolar (antiparallel) minifilament that has motor domains at both ends of the filament. This homotetramer has a "slow" motor activity that moves microtubules at 0.04 microns/second in an in vitro motility assay (4). Members of the kinesin II family are heterotrimers composed of two distinct motor molecules (~85 and 95 kDa) and a third noncatalytic subunit (~120kDa) in a 1:1:1 ratio. These molecules move protein complexes, "rafts," along the inner surface of the flagellar membrane and are important for flagellar function (5). The Kar3 family of kinesins has motor domains at the carboxy terminus of the heavy chain, and they are the exception to the rule that kinesins move toward the plus ends of microtubules. They generate force toward the minus ends of microtubules. These proteins are required for forming and maintaining the meiotic and mitotic spindles. How the Kar3 head domain moves in the opposite direction along microtubules compared to the other kinesin head domains is under investigation (6).
