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Review Molecular Analysis of the Microtubule Motor Dynein Richard Vallee Cell Biology Group, Worcester Foundaton for Eerimental Biology, Shrwsury, MA 01545

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Review Molecular Analysis of the Microtubule Motor Dynein Richard Vallee Cell Biology Group, Worcester Foundaton for Eerimental Biology, Shrwsury, MA 01545 Proc. Natl. Acad. Sci. USA Vol. 90, pp. 8769-8772, October 1993 Review Molecular analysis of the microtubule motor dynein Richard Vallee Cell Biology Group, Worcester Foundaton for Eerimental Biology, Shrwsury, MA 01545 ABSTRACT Dynein is a large enzyme remainder ofthe heavy chain is predicted Molecular Cloning of Dynein complex that has been found in recent to form a coiled-coil a-helix (12-15). This Heavy Chain years to be responsible for a variety of arrangement is strikingly reminiscent of forms of intracellular movement associ- that of muscle myosin, though, other Heavy chains from sea urchin flagellar ated with microtubules. Molecular analy- than the presence ofthe P-loop within the dynein (28, 29) and from Dictyostelium sis of several of the polypeptide compo- head domain, no evidence for primary (30) and rat (microtubule-associated pro- nents of dynein and a related complex has sequence homology with myosin was de- tein 1C; ref. 31) cytoplasmic dynein have provided important new insight into their tected. In further contrast to myosin, the been fully cloned and sequenced. Full- structural organization and mechanism of kinesin light chains (16) are associated length sequences are near completion for action in the cell. with the C-terminal tail portion of the two of the three different forms of Chla- molecule rather than the heads. As for mydomonas flagellar dynein heavy chain Within the past decade cytoplasmic mo- myosin (reviewed in refs. 17-20), afamily (C. Wilkerson, S. King, and G. Witman, tor proteins have been identified that of kinesin-related genes has been identi- personal communication; D. R. Mitchell produce force along microtubules in vi- fied, which exhibit clear sequence con- and K. Brown, personal communication) tro. These proteins are microtubule- servation within the head region and con- and for Caenorhabditis elegans (32) and activated ATPases, which convert chem- siderable variation within the remainder Saccharomyces cerevisiae (33, 34) cyto- ical energy into mechanical energy. Ki- of the molecule and have roles in a vari- plasmic dyneins. In addition, sequences nesin was found to generate force toward ety of forms of intracellular movement from the catalytic domains of several the plus ends of microtubules and was (reviewed in refs. 21-23). other forms ofdynein have been obtained proposed to account for anterograde ax- Dyneins are much larger and more by PCR from sea urchin (35), Drosophila onal transport and other forms of plus- complex molecules than the several (K. Rasmussen, J. Gepner, M. Serr, I. end-directed organelle movement along forms of myosin or kinesin (molecular Gibbons, and T. Hays, personal commu- microtubules (1). Cytoplasmic dynein mass <600 kDa) that have been identi- nication), Chlamydomonas (36, 37), and was identified as a minus-end-directed fied. Dyneins have a native mass be- Paramecium (38). The predicted sizes of counterpart of kinesin and was proposed tween 1000 and 2000 kDa and contain the completed heavy chains are in the to account for retrograde, or minus-end- either two or three force-producing range of 510-540 kDa. Based on the directed, movements along microtubules heads, each ofwhich is about as massive pattern of expression of the different (2). It was subsequently shown to be as the entire kinesin molecule (3, 24, 25). forms of heavy chain, genomic Southern structurally (3) and biochemically (4-6) The heads are linked via stalks to a basal blot analysis, and parsimony analysis of related to the ciliary and flagellar ATPase domain, which is as large as the heads but the evolutionary relationship between dynein. In cilia and flagella, dynein is less well defined structurally. The heads the different forms of heavy chain, it responsible for generating force between are thought to be formed primarily or appears that organisms generally have a the array of20 microtubules that make up exclusively from the heavy chains, ex- single cytoplasmic dynein heavy chain the characteristic "axonemal" structure. tremely large polypeptides responsible gene and numerous axonemal dynein Cytoplasmic dynein, in contrast, has for ATP hydrolysis (see below). In addi- heavy chain genes (30, 31, 35). The latter been implicated in a variety of other are thought to reflect the multiplicity of tion, dyneins contain a highly variable within forms of intracellular motility (reviewed number of accessory intermediate and heavy chain polypeptides found in refs. 7-9), including retrograde axonal contain a individual axonemal dynein molecules transport, protein sorting between the light chains. Axonemal dyneins (the Chlamydomonas outer arm dynein apical and basolateral surfaces of epithe- variety of such subunits ranging in size molecule is three-headed and contains lial cells, and the distribution and redis- from 414 to 120 kDa (26). Cytoplasmic three distinct heavy chain gene products; tribution of endosomes, lysosomes, and dynein contains at least seven different refs. 39 and 40) and among the several the elements of the Golgi apparatus accessory polypeptides ranging from 53 forms of dynein observed within a given within the cell. Cytoplasmic dynein has to 74 kDa (3, 4). In addition, a number of axoneme (one outer arm dynein and at also been implicated in the poleward mi- partially copurifying polypeptides have least three different inner dynein arms; gration of chromosomes, at least during been identified that may be involved in refs. 41-43). Overall sequence identity some stages of mitosis. regulating cytoplasmic dynein behavior between cytoplasmic and axonemal dy- Substantial information is already (see below). neins can be as low as 27% (rat cytoplas- available regarding the structural organi- Because ofthe large size ofthe dyneins mic dynein vs. sea urchin axonemal dy- zation of kinesin based on primary se- and their complex composition, defining nein), whereas the degree of conserva- quence, physicochemical, and ultrastruc- their structure has been a daunting un- tion between species for the same tural analysis. The molecule is a tetramer dertaking. Nonetheless, recent progress functional form of dynein tends to be of two heavy and two light chains (10, in the molecular characterization of their higher (for example, 54% between rat 11). The N-terminal 35 kDa of the heavy component polypeptides has provided cytoplasmic and Dictyostelium cytoplas- chain contains a P-loop consensus se- new insight into their structural organi- mic dynein; see ref. 31). Little evidence quence element indicative of nucleotide zation and mechanism of action. This for significant homology with the kine- binding and hydrolysis and represents work is reviewed here and in expanded sins and myosins was detected (though the force-producing "head" domain. The form elsewhere (27). see ref. 44). 8769 Downloaded by guest on October 2, 2021 8770 Review: Vallee Proc. Natl. Acad. Sci. USA 90 (1993) The most remarkable common features *- Variable - 0 Conserved of the several dynein heavy chain se- quences are four P-loop consensus se- ATP-binding quence elements spaced at 35- to 40-kDa intervals (Fig. 1). The region surrounding .................::.....:: .:..:.:..... the first P-loop represents the most highly I I I conserved part of the molecule. UV- 0 1000 2000 3000 4000 induced cleavage of dynein heavy chains FIG. 1. Diagram of dynein heavy chain. White rectangles represent phosphate-binding in the presence ofvanadate, which results P-loop sequence elements; the shaded region differs between known complete axonemal (sea in the inactivation ofthe ATPase activity, urchin flagellar) and cytoplasmic dynein heavy chain sequences (rat and Dictyostelium). is estimated to occur in the vicinity ofthis site (28), suggesting an involvement in primary sequence has offered relatively sequences near the 5' end, suggesting ATP hydrolysis. The third P-loop region is little in the way ofinsight into the tertiary that the multiple 74-kDa electrophoretic also relatively well conserved among dy- structural organization of the molecule. species were produced by an alternative neins and most similar to the first P-loop The large head domain probably reflects splicing mechanism. More recently, sequence. This suggests that the four do- the multiplicity of ATP-binding regions cDNAs encoding the 78-kDa Chlamy- mains may have arisen during evolution (Fig. 2A), but the requirement for this domonas dynein subunit (IC78) were by successive duplications of the first se- level of structural redundancy remains cloned and sequenced revealing a similar quence and then of the first and second obscure. pattern of homology with both the sequence. Such duplications would pre- Chlamydomonas flagellar and the 74-kDa sumably be very ancient, appearing as Molecular Cloning of Dynein rat cytoplasmic dynein subunits (51). they do in all forms ofdynein heavy chain. Accessory Subunits These results provide further evidence Whether the second, third, and fourth for the common ancestry of axonemal P-loop elements are vestigial or serve a Progress has also been made in the mo- and cytoplasmic dyneins and identify a regulatory role remains to be investigated. lecular cloning of the dynein accessory novel intermediate chain gene family. Kinetic evidence has been interpreted to which has some in- support binding ofonly one ATP molecule subunits, provided These findings, in conjunction with per heavy chain (45). However, because sight into their functional relationship. structural and functional studies of the of the complexity of the dynein holoen- The Chlamydomonas flagellar outer axonemal dynein intermediate chains, zyme (which contained three heavy arm dynein, which has been one of the have suggested a role for the 74-kDa chains in the study cited), detailed kinetic most extensively studied forms of the cytoplasmic dynein subunit. Axonemal analysis of recombinant heavy chain or enzyme, contains three distinct heavy and cytoplasmic forms of dynein are pre- other simplerforms ofthe molecule seems chains, two intermediate chains of70 and sumed to produce force via a common warranted. 78 kDa, and a series of light chains in the mechanism involving their conserved Clear sequence conservation between 10- to 30-kDa range (39).
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