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Proc. Natl. Acad. Sci. USA Vol. 90, pp. 8769-8772, October 1993 Review Molecular analysis of the motor Richard Vallee Biology Group, Worcester Foundaton for Eerimental Biology, Shrwsury, MA 01545

ABSTRACT Dynein is a large 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 , though, other Heavy chains from sea urchin flagellar ated with . 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 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 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 have been identified that of kinesin-related 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 (32) and activated , 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 . 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), onal transport and other forms of plus- complex molecules than the several (K. Rasmussen, J. Gepner, M. Serr, I. end-directed 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 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 (reviewed number of accessory intermediate and heavy chain polypeptides found in refs. 7-9), including retrograde axonal contain a individual axonemal dynein molecules transport, 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 , , and dynein contains at least seven different refs. 39 and 40) and among the several the elements of the 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 (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 , at least during been identified that may be involved in refs. 41-43). Overall sequence identity some stages of . 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 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 of dynein heavy chains FIG. 1. Diagram of dynein heavy chain. White rectangles represent -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). Purified rat head domains. However, they differ in axonemal and cytoplasmic dynein heavy cytoplasmic dynein contains a prominent the nature oftheir additional interactions chains extends over the C-terminal two- polypeptide of 74 kDa, which has been within the cell, which are thought to be thirds of the polypeptides (30, 31). How- observed to split into as many as three mediated by the basal portion of the ever, the N-terminal 1300 amino acids electrophoretic bands, and additional molecule (Fig. 2B). In the case of axone- appear to be completely unrelated be- polypeptides of59, 57, 55, and 53 kDa (3, mal dynein, this part of the molecule tween the flagellar and cytoplasmic se- 4). The differences in subunit composi- forms a fixed attachment to a second quences. In contrast, the rat and Dicty- tion between the two dynein forms have microtubule within the axoneme, which ostelium sequences show 43% sequence made it difficult to judge to what extent results in sliding between the axonemal identity over this region (31), suggesting individual polypeptides are structurally microtubules and bending of the entire a role in cytoplasmic- and axonemal- or functionally related. or . The base of the cy- specific functions. Because the predicted Molecular cloning of the 70-kDa toplasmic dynein molecule is predicted to masses of the heavy chains are much Chlamydomonas flagellar dynein outer form a comparable attachment to the greater than the observed masses of the arm intermediate chain (IC70) was ac- surface of membranous and, force-producing head domains [327 kDa complished in conjunction with the anal- possibly, . for cytoplasmic dynein (3) and 375-400 ysis of outer dynein arm (oda) mutants Both IC70 and IC78 of Chlamydomo- kDa for axonemal dyneins (24, 46)], ei- (47). Several of these mutants, including nas flagellar outer arm dynein have been ther or both the N- and C-terminal por- oda6, lack outer arms completely as deduced to reside in the basal portion of tions ofthe heavy chain may lie outside of judged morphologically and biochemi- the molecule, as judged by immunoelec- the head domain and contribute to the cally, and show abnormal flagellar motil- tron microscopic analysis of purified dy- stalk and basal portions of the dynein ity (48). cDNA clones encoding the 70- nein particles using an anti-IC70 molecule. Conceivably, the noncon- kDa intermediate chain (49) were shown (52) and biochemical evidence showing a served N-terminal domain specifies cy- to map to the ODA6 locus and were used direct interaction between IC70 and IC78 toplasmic vs. axonemal function, playing to deduce the primary sequence of its (53). Cross-linking studies revealed a di- a role either in subunit interactions or in polypeptide product (47). rect interaction of the 78-kDa species targeting the enzyme to distinct subcel- Cloning and sequencing of overlapping with (54), as did binding of the in lular sites (Fig. 2). cDNAs encoding the rat cytoplasmic dy- vitro-translated polypeptide to purified While molecular cloning of the dynein nein 74-kDa species revealed clear, albeit microtubules (51). These data suggest heavy chains has provided a basis for relatively distant, homology with the that the role of IC78, and possibly of further exploration of the relationship Chlamydomonas IC70 (50). Sequence IC70, is in producing the noncatalytic between dynein structure and function, it conservation was detected primarily link to microtubules (Fig. 2). has also raised many questions. There is within the C-terminal portion of the two An analogous role for the 74-kDa cyto- still no indication as to why the dynein sequences. Heterogeneity among 74-kDa plasmic dynein subunit would be in bind- heavy chain is so large (the head alone is transcripts indicated by PCR analysis and ing to organelles and kinetochores (Fig. much larger than the 100-kDa tubulin sequencing ofproteolytic frag- 2B), but the details of this interaction are dimer with which it interacts), and the ments revealed at least three alternative poorly understood. Cytoplasmic dynein, Downloaded by guest on October 2, 2021 MReview: Vallee Proc. Natl. Acad. Sci. USA 90 (1993) 8771 AB The 45-kDa species, which is the major component ofthe complex, also proved to have an interesting identity. Peptide se- quence from both the chicken (63) and rat (62) polypeptides revealed them to repre- sent a member of a family ofactin-related proteins, referred to as centractin (64) or actinRPV (63). The 34- and 32-kDa poly- peptides have been identified immunolog- ically as the capping protein CapZ (65). Immunoblotting ofsucrose gradients of brain indicated the three major components of the complex, p150GIuIId, (Organelle, , p50, and centractin to exist exclusively in Microtubule) a 20S form (62), a surprising result con- FIG. 2. (A) Sketch of heavy chain, indicating hypothetical folding pattern. The P loops are sidering the polymorphic nature of actin. speculated to demarcate repeated structural domains of35-40 kDa. While the first such domain While the complex is incompletely sep- is thought to be involved in catalytic activity, the role of the other domains is uncertain. The arated from cytoplasmic dynein by FPLC C- and N-terminal portions of the heavy chain are speculated to make up the stalk of the (61), complete separation was seen by molecule. (B) Interaction of dynein with microtubules and other structures. Dynein heads, immunoprecipitation (62), raising the is- representing ATPase region ofheavy chains (HC), interact with a microtubule at top to produce sue ofwhether the two structures interact force. Intermediate chains (ICs) are shown attached to the basal region ofthe dynein molecule, at all in vitro or in vivo. where they interact with a second microtubule in cilia and flagella or with organelles and The evidence in favor of such an inter- possibly kinetochores in the . (Adapted from ref. 27.) action is intriguing but incomplete. Cyto- like kinesin, is found at a substantial con- Role of the Glued () Complex plasmic dynein alone was found to have centration in the soluble phase of tissue no effect on microtubule-associated or- homogenates, with little evidence for re- In addition to the biochemically well- ganelle movements in an in vitro assay sidual enzyme. using organelles stripped of peripheral membrane-associated behaved components of cytoplasmic dy- membrane proteins (66). However, addi- Whether this indicates a weak affinity for nein described above, a number of par- tion ofthe complex stimulated the number membrane binding sites or a regulated tially copurifying polypeptides have been of organelle movements per unit time. interaction is not known. No evidence for seen, including species of 150, 135, 50, Immunocytochemical analysis using a membrane-spanning domain in the se- and 45 kDa (59-61). These polypeptides to the components ofthe com- quence ofthe 74-kDa subunits was found, cosediment with microtubules, though plex has revealed a punctate cytoplasmic and sequence motifs clearly indicative of less efficiently than the heavy chain and distribution, which may correspond to lipid modification were not identified. It 74-kDa intermediate chains of cytoplas- vesicular structures, with particularly seems likely, therefore, that the cytoplas- mic dynein; they dissociate from micro- bright staining at the [refs. mic dynein subunit will prove to interact tubules in the presence ofATP along with 61, 62, and 64; and hence the name cen- with other polypeptides on the surface of cytoplasmic dynein; and they cosediment tractin for the actin-related component organelles and kinetochores. with cytoplasmic dynein at 20 S (61, 62). (64)]. The latter distribution can be dis- Immunocytochemical analysis has re- However, the four polypeptides, along rupted using microtubule depolymerizing vealed a clear association of cytoplasmic with additional minor species of 62, 34, drugs (62). This suggests that the com- dynein with two classes of membranous and 32 kDa, can be separated from cyto- plex is associated with the centrosome organelles, lysosomes and late endosomes plasmic dynein by FPLC (61). Antibody peripherally via the minus ends ofmicro- (55). These organelles often exhibit a peri- to the 50-kDa polypeptide has also been tubules, which are anchored there. The nuclear consistent with a role distribution, found to immunoprecipitate the same immunocytochemical behavior of the for a minus-end-directed microtubule mo- components in a comparable ratio from components of the complex is consistent tor in controlling their subcellular distri- with that expected for a cytoplasmic have total brain cytosol (45 kDa > 50 kDa > bution. While anti-dynein antibodies 150 kDa > 135 kDa >> 62 kDa > 32 kDa structure under the spatial control of cy- not shown comparable so toplasmic dynein. Curiously, however, Golgi staining > 34 kDa). Together, these data reveal far, dynein may be associated with this while lysosomes, endosomes, and the these polypeptides to be components of a organelle as well. Golgi membranes intro- Golgi apparatus are often found in the duced into broken cell preparations have discrete complex (62). pericentrosomal region, they are not been reported to become localized to the Molecular cloning of the 150-kDa spe- sharply focused at the centrosome itself. centrosomal region, and this behavior was cies in rat brain revealed it to be homol- Thus, the complex may be directly asso- abolished by immunodepletion of cyto- ogous throughout its length to the similar- ciated with the microtubule minus ends, plasmic dynein (56). sized product of the Glued gene in Dro- or it may serve as a marker for an, as yet, While direct binding of cytoplasmic sophila (60). A cDNA encoding a poorly characterized vesicular compart- dynein to purified minus-end-directed or- polypeptide corresponding to the C-ter- ment that resides in this region. ganelles in vitro has not been assessed, minal 117 kDa of the rat and fly polypep- Evidence for a specific interaction of binding to synaptic vesicles (57) and mi- tides was subsequently reported under the the complex with microtubules has also crosomes (58) has been reported. It will name dynactin (61). (The complex has come from work on two other proteins, be of interest to determine whether cy- come to be referred to by the names Glued CLIP170 and BIK1. CLIP170 copurifies toplasmic dynein will discriminate be- or dynactin.) The original Glued mutant is with and colocalizes with both microtu- tween different classes of organelles in in dominant and produces defects in the de- bules and endosomes, implicating the pro- vitro binding assays, and it should be of velopment ofthe eye and . tein in cross-linking the two structures value to identify organelle and kineto- Homozygotes have a cell lethal pheno- (67). An N-terminal repeated sequence of chore surface proteins with which the type, indicating a role in an essential cell =100 amino acids was found to be respon- 74-kDa cytoplasmic dynein intermediate function, but no information is available sible for microtubule binding in vitro. chain interacts. identifying a specific cellular defect. BIKi (68, 69) is a yeast gene, the product Downloaded by guest on October 2, 2021 8772 Review: Vallee Proc. Natl. Acad. Sci. USA 90 (1993)

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