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membranes and pigment granules. Mitotic kinesins, in contrast, participate in spindle assembly and chromosome segregation in cell division. This group comprises numerous proteins, including the kinetochore-associated protein CENP-E, the bipolar BimC, and a (—) end-directed motor protein called Ncd. The functions of mitotic kinesins are described in more detail in Section 20.3.

A sequence called the tetratrico peptide sequence has been recently identified in the light chains of kinesin I and may interact with receptor proteins in the membrane of various cargoes. Such interactions would tether the cargo organelle or vesicle to kinesin. For instance, the tetratrico peptide sequence has been found to bind to several proteins, including the amyloid precursor protein. Other kinesins may have different interaction sequences that bind to other receptors on membranes.

Two fundamental properties of the kinesin motor—its step size and force—have been determined in optical-trap experiments similar to those performed on myosin (see Figure 19-18). Findings from these studies show that a dimeric kinesin molecule (e.g., kinesin I) exerts a force of 6 piconewtons, which is sufficient to pull a bound vesicle through the viscous cytoplasm. The kinesin step size of 8 nm matches the distance between successive a- or ^-tubulin monomers in a protofilament, suggesting that kinesin binds to only one or the other monomer. Electron microscopy reconstructions show that kinesin binds primarily to ^-tubulin. In other experiments, researchers have established that a double-headed kinesin molecule moves along a single protofilament, with one head always bound to the mi-crotubule. As a result, a kinesin molecule can move along a mi-

crotubule for a long distance without detaching from it, a property referred to as processivity. Because of their high processiv-ity, dimeric kinesins are very efficient in transporting cargo from one part of a cell to another.

In Chapter 3, we saw that the neck region of myosin, which acts as a rigid lever arm, is critical in coupling ATP hydrolysis to the movement of myosin along an actin microfilament. In contrast with myosin, kinesin has a flexible neck domain, which links the head domain to the central stalk domain (see Figure 20-20). Current models propose that ATP hydrolysis by kinesin causes movement of the flexible neck, which then positions the head domain into the next step along a microtubule protofilament. According to this model, the direction of kinesin movement depends on neck function, not on the motor domain. This function of the neck is supported by findings from recent domain-replacement experiments. For example, replacing the motor domain in (—) end-directed Ncd with the motor domain from (+) end-directed kinesin I yielded a (—) end-directed chimeric protein. Likewise, swapping the kinesin and Vcd motor domains into kinesin I produced a (+ ) end-directed protein. These results show that the direction of movement is not an intrinsic property of the motor domain. However, mutations in the neck region of Ncd converted it from a (— ) into a (+ ) end-directed motor protein.

Cytosolic Dyneins Are (—) End-Directed Motor Proteins That Bind Cargo Through Dynactin

The second family of microtubule motor proteins, the dyneins, is responsible for retrograde axonal transport,

▲ FIGURE 20-22 Cytosolic dynein and the dynactin heterocomplex. (a) Diagram of dynein (green) bound to the dynactin complex (orange) through interactions between the dynein light chains and the dynamtin subunits of dynactin. The Arp1 subunits of dynactin form a minifilament that associates with spectrin underlying the cell membrane. The Glued subunits bind microtubules and vesicles. (b) Electron micrograph of a metal replica of the dynactin complex isolated from brain cells. The Arp1 minifilament (purple) and the dynamtin/Glued side arm (blue) are visible. [Part (a) adapted from N. Hirokawa, 1998, Science 279:518. Part (b) from D. M. Eckley et al., 1999, J. Cell Biol. 147:307.]

transit of Golgi vesicles to the centrosome, and some other (—) end-directed movements. Dyneins are exceptionally large, multimeric proteins, with molecular weights exceeding 1 X 106. They are composed of two or three heavy chains complexed with a poorly determined number of intermediate and light chains. As summarized in Table 20-2, the dyneins are divided into two functional classes. Here we consider cytosolic dynein, which has a role in the movement of vesicles and chromosomes. Axonemal dyneins, responsible for the beating of cilia and flagella, are considered later.

Like kinesin I, cytosolic dynein is a two-headed molecule, with two identical or nearly identical heavy chains forming the head domains. However, unlike kinesin, dynein cannot mediate cargo transport by itself. Rather, dynein-related transport requires dynactin, a large protein complex that links vesicles and chromosomes to the dynein light chains (Figure 20-22). The results of in vitro binding experiments show that dynactin also binds to microtubules, thereby enhancing the processivity of dynein-dependent movement. Dynactin consists of at least eight subunits, including a protein called Glued, which binds microtubules; Arp1, an actin-related protein that binds spectrin; and dynamtin, which interacts with the light chains of dynein. The microtubule-binding site in Glued contains a 57-residue motif that is also present in CLIP170, a microtubule-associated protein that cross-links microtubules and endocytic vesicles (see Table 20-1). One model proposes that dynein generates the force for vesicle movement but remains tethered to a microtubule through dynactin.

As discussed later, several lines of evidence suggest that the dynein-dynactin complex and another complex, the nu-clear/mitotic apparatus (NuMA) protein, mediate the association of microtubules with centrosomes in mitosis. The results of in vitro studies show that truncated NuMA protein binds microtubules if the C-terminal region is retained. As in MAPs, the C-terminal region of NuMA protein is highly acidic, and ionic interactions may mediate its binding to microtubules.

Multiple Motor Proteins Sometimes Move the Same Cargo

Figure 20-23 summarizes the role of kinesins and cytosolic dyneins in intracellular transport along microtubules. Because the orientation of microtubules is fixed by the MTOC, the direction of transport—toward or away from the cell periphery—depends on the motor protein. Some cargoes, such as pigment granules, can alternate their direction of movement along a single microtubule. In this case, both an-terograde and retrograde microtubule motor proteins must associate with the same cargo. Recent biochemical experiments have identified dynactin in a complex with kinesin. A model proposes that dynactin is part of the membrane receptor and serves as a common adapter for binding kinesin and cytoplasmic dynein. Thus the direction of movement can be switched by swapping one motor protein for the other.

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▲ FIGURE 20-23 General model of kinesin- and dynein-mediated transport in a typical cell. The array of microtubules, with their (+) ends pointing toward the cell periphery, radiates from an MTOC in the Golgi region. Kinesin-dependent anterograde transport (red)

conveys mitochondria, lysosomes, and an assortment of vesicles to the endoplasmic reticulum (ER) or cell periphery. Cytosolic dynein-dependent retrograde transport (green) conveys mitochondria, elements of the ER, and late endosomes to the cell center. [Adapted from N. Hirokawa, 1998, Science 279:518.]

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Cell membrane

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