Journal of Structural Biology 156 (2006) 175–181

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Autoinhibitory and other autoregulatory elements within the dynein motor domain

Richard B. Vallee ¤, Peter Höök

Department of Pathology and Cell Biology, Columbia University College of Physicians and Surgeons, P and S 15-410, 630 W. 168th St., New York, NY 10032, USA

Received 23 December 2005; received in revised form 2 February 2006; accepted 17 February 2006Available online 29 March 2006

Abstract

The dyneins are a family of microtubule motor proteins. The motor domain, which represents the C-terminal 2/3 of the dynein heavychain, exhibits homology to the AAA family of ATPases. It consists of a ring of six related but divergent AAA+ units, with two substan-tial sized protruding projections, the stem, or tail, which anchors the protein to diverse subcellular sites, and the stalk, which binds micro-tubules. This article reviews recent eVorts to probe the mechanism by which the dyneins produce force, and work from the authors’ labregarding long-range conformational regulation of dynein enzymatic activity.© 2006 Elsevier Inc. All rights reserved.

Keywords: Dynein; aaa protein; aaa domain; ATPase; ATPase activity; Motor protein; Stalk

1. Dynein superfamily

The dyneins are a subfamily of the AAA+ proteins. Twogeneral forms of dynein are found in eukaryotes. The axo-nemal dyneins are associated with the bundle of microtu-bules—the axoneme—comprising the cilium or Xagellumand are responsible for ciliary and Xagellar beating. Thehuman genome contains 13 forms of axonemal dynein. Sur-prisingly, this degree of complexity in large part reXects themultiplicity of dyneins functioning within a given axoneme,the activities of which are coordinated to produce highlyspeciWc wave forms (Höök and Vallee, in preparation; Por-ter, 1996).

There are two forms of cytoplasmic dynein. The majorform, also referred to as MAP1C or dynein 1, is ubiqui-tously expressed (Paschal et al., 1987; Paschal and Vallee,1987). In contrast to the axonemal dyneins, this one form ofdynein is involved in a very wide range of cellular functions(Vale, 2003; Vallee et al., 2004). It associates with diverse

* Corresponding author. Fax: +1 212 305 5498.E-mail address: rv2025@columbia.edu (R.B. Vallee).

1047-8477/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.jsb.2006.02.012

membranous organelles, including the Golgi apparatus andcomponents of the endosomal/lysosomal pathway, which ittransports toward the microtubule minus end. During theearly stages of mitosis, the same form of dynein appears atmitotic kinetochores. Its role at these sites is incompletelyunderstood, though it may participate in the initial captureof chromosomes by microtubules and in removing check-point proteins at the onset of anaphase. Dynein 1 alsoappears at the cell cortex, from which site it is thought toorient the mitotic spindle. In migrating cells, it appears atthe leading cell cortex, from which site it orients the micro-tubule cytoskeleton and may participate in cell steering.Finally, dynein 1 has been implicated in the transport ofdiverse macromolecular complexes, including mRNA com-plexes, centrosomal precursor complexes, and a number ofviruses.

A second form of cytoplasmic dynein, known as dynein2, is much less abundant in cells and has a more restrictedcell type distribution (Grissom et al., 2002; Mikami et al.,2002; Pazour et al., 1999; Perrone et al., 2003; Porter et al.,1999; Signor et al., 1999). Curiously, it is found primarilyassociated with cilia and Xagella. However, unlike the axo-nemal dyneins, dynein 2 has been implicated in transport of

176 R.B. Vallee, P. Höök / Journal of Structural Biology 156 (2006) 175–181

materials within the space between the outer surface of theaxoneme and the surrounding ciliary or Xagellar plasmamembrane.

The dyneins are responsible for a very broad range ofsubcellular functions. They are fast motors, responsible formovements in the 1–10�m/s range, generally much fasterthan the members of the kinesin protein superfamily. Infurther contrast to the kinesins, the dyneins are unusuallylarge proteins, and the dynein motor domain alone is tentimes the size of the kinesin motor. For this reason, pro-gress in elucidating the detailed mechanism for dynein forceproduction has progressed more slowly.

2. The dyneins as divergent members of the AAA+ family

Each of the dyneins is a multisubunit complex. Themajor functional subunit is the heavy chain (Fig. 1), whichranges in size between 470 and 540 kDa. The cytoplasmicdyneins are homodimers of the heavy chain, and containdiverse accessory subunits (Mikami et al., 2002; Perroneet al., 2003; Vallee et al., 1988). Axonemal dyneins containone heavy chain, two non-identical heavy chains, or, inlower eukaryotes, three nonidentical heavy chains (Goo-denough et al., 1987; Porter, 1996; Sakakibara et al., 1999).

The dynein heavy chains are responsible for ATPase andmotor activity. The motor domain, alone, represents »2/3of the mass of the heavy chain, some 350–380 kDa. Eachmotor domain contains six identiWable AAA units (Neu-wald et al., 1999). These are more highly conserved betweenspecies than within the same heavy chain polypeptide. Thisaspect of dynein evolution, as well as the conservation ofother structural features indicates that the organizationalplan of the dynein heavy chain is very ancient.

The dynein AAA units are organized into a ring (Bur-gess et al., 2003; Samso et al., 1998), a general characteristicof proteins in the AAA+ superfamily. The motor domaincontains at least two additional identiWable structures. The“stalk” is a 10–15 nm long extension located betweenAAA4 and AAA5 (Gee et al., 1997; Goodenough andHeuser, 1984). The shaft of the stalk is thought to consist acoiled-coil �-helix, based on secondary structure predic-tions (Gee et al., 1997). Because the coiled-coil must beintrachain, it is likely to have an antiparallel organization,though this model has not been tested directly. At the tip ofthe stalk is a small globular domain that is responsible formicrotubule binding. How ATP hydrolysis within the AAAdomains cycle is coupled to and coordinated with microtu-bule binding represents one of the more important andintriguing mysteries of the Weld.

The N-terminal 1/3 of the heavy chain represents thestem of the dynein molecule. This region anchors cytoplas-mic dyneins to subcellular forms of cargo, such as Golgimembranes and kinetochores, and axonemal dyneins to theA microtubule in each pair of outer doublet microtubules.The initial 1100 amino acids are sites for heavy chaindimerization and binding of accessory subunits (the inter-mediate, light intermediate, and light chains) (Habura et al.,1999; Tynan et al., 2000). The 600 or so amino acidsbetween residues 1300 and 1900, and the N-terminalboundary of AAA1 appears to represent an additionalfunctional part of the motor. This conclusion is based inpart on deletion analysis (Gee et al., 1997), which showedthat N-terminal truncations which removed portions of thisdomain interfered with one indicator of enzymatic activityat AAA1, VO4-mediated photocleavage (see below). Thisregion also appears to correspond to a structural feature

Fig. 1. Diagrammatic representation of the dynein heavy chain and its recombinant and proteolytic fragments. The full-length dynein heavy chain isshown at top. Precise boundaries of the linker domain are uncertain. Complete dynein motor domain (380 kDa fragment) is depicted as ring of AAAdomains and C-terminal domain. Stalk is shown projecting from between AAA4 and AAA5. Portion of linker protruding from ring is shown in blue.Junction between linker and AAA remains incompletely deWned. The 210-kDa fragment consists of linker-AAA4. Tryptic sites 1 and 2 are almost identicalbetween 380 and 210-kDa motor polypeptides. Tryptic site 3 is eliminated in presence of enzymatic transition-state analogues. Adapted from (Höök et al.,2005).

Stem (Linker) AAA1 AAA2 AAA3 AAA4 Stalk AAA5 AAA6 CT

Trypsin Trypsin Trypsin

380 kDa

Trypsin Trypsin

210 kDa

R.B. Vallee, P. Höök / Journal of Structural Biology 156 (2006) 175–181 177

termed the “linker” (Burgess et al., 2003), the existence ofwhich has been deduced from recent electron microscopicanalysis (see below) and which may play a critical role inthe dynein power stroke.

3. Models for motor function

How the dynein motor domain functions to produceforce is poorly understood. Unlike multimeric AAA pro-teins, the AAA units within the dyneins diVer from eachother functionally. AAA1, AAA2, and AAA3 have wellconserved Walker A motifs (Neuwald et al., 1999). Thismotif is conserved in AAA4, but only in some species, andabsent in AAA5 and AAA6 (Höök and Vallee, in prepara-tion). The Walker A motifs in AAA1–4 were recognizablebefore the relationship of the dynein heavy chain to theAAA family was appreciated, and the Wrst Walker A motifwas understood as functionally important (Gibbons et al.,1991). This conclusion was based on VO4-mediated photo-cleavage experiments, which took advantage of the highaYnity of dyneins for ADP-VO4 and the ability of VO4 toabsorb light in the UV region (Gibbons et al., 1987). Illumi-nation of dynein-bound VO4 induced scission of the dyneinheavy chain in the vicinity of the Wrst Walker A motif. Theresults were inactivation of dynein and almost completeloss of ATPase activity.

Subsequent mutational analysis has indicated thatAAA1 and AAA3 are both required for dynein activity(Kon et al., 2004; Reck-Peterson and Vale, 2004; Silvano-vich et al., 2003). Mutations in the Walker A motif ofAAA1 and AAA3, each interfered with microtubule-acti-vated ATPase activity as well as ATP-induced release frommicrotubules, whereas mutations in AAA2 and AAA4 hadno eVect on these aspects of dynein behavior.

How the dynein motor domain converts the energyreleased by ATP hydrolysis to produce force, has been anissue of intense recent interest. Single particle image averag-ing of motor domains visualized by electron microscopyhas revealed a ring-shaped structure, consistent with otherAAA family members (Samso et al., 1998). Seven globularsubdomains were detected, presumably corresponding tothe AAA units plus, perhaps, an additional C-terminaldomain (see below). Similar analysis of a complete, singleheaded Xagellar dynein, dynein c of Chlamydomonas rein-hardtii, revealed further detail, including evidence for a hid-den connector—the linker (Burgess et al., 2003). Averagingof subsets of images allowed the stalk and stem of the mole-cule to be imaged with considerable clarity. Evidence for amissing segment situated between AAA1 and the stem por-tion of the molecule was indirect, but compelling. In mostmolecules, the stalk and stem emerged from the ring quiteclose to each other. This result contradicted expectationsbased on the domain organization of the heavy chain(Fig. 1), and suggested that a portion of the dynein mole-cule must in some way be masked by the AAA ring. In sup-port of this possibility, the hole in the ring was occluded ina number of views. Furthermore, the stem appeared elon-

gated by some 10 nm in occasional views. In these cases, thestem was seen to emerge from the ring opposite to the stalk.Together these results led to the proposal that the N-termi-nal-most portion of the motor domain is twisted in such away as to interact with the surface of the AAA ring.

Molecules prepared in the nucleotide-free versus theADP-VO4 bound state, representing the post- and pre-power stroke states, showed a substantial diVerence in con-formation, corresponding to a step along the microtubulesurface of »15 nm. Much of this change was attributed to ashift in the interaction between the linker and the ring.FRET analysis using GFP and BFP motor domain fusionshas been reported to be consistent with movement of thelinker relative to the AAA ring (Kon et al., 2005).

The role of the stalk remains to be fully elucidated. Thishighly unusual domain protrudes some 10–15 nm from theAAA ring (Goodenough and Heuser, 1984). Microtubulebinding activity is associated with a 125 a.a. globulardomain at the tip of the stalk, which is bracketed by tworegions predicted coiled-coil �-helical structure. Whetherthe stalk contributes to the dynein power stroke is uncer-tain. How the interaction of the stalk tip with the microtu-bules regulates dynein ATP hydrolysis and vice versaremains largely unexplored.

4. Evidence from recombinant dynein fragments for stalk regulation of product release

Our lab has recently investigated intramolecular regula-tion of dynein enzymatic activity by analysis of recombi-nant and proteolytic motor fragments (Höök et al., 2005).Using baculovirus infection of insect cells, we expressed a380-kDa fragment corresponding to the entire motordomain and a 210-kDa fragment corresponding to its N-terminal half, ending just prior to the stalk (Fig. 1). Thisjunction corresponds closely to the natural boundarybetween two heavy chain fragments expressed indepen-dently and assembled into a functional motor subunit inthe corn smut Ustilago maydis (Straube et al., 2001). Boththe 380 and 210 kDa baculovirus-expressed dynein motorfragments were soluble and reasonably stable enzymati-cally.

The steady-stage ATPase activity of the complete motordomain, which had been cloned from rat, correspondedwell with values obtained for the vertebrate cytoplasmicdynein holoenzyme (Paschal et al., 1987; Shpetner et al.,1988). Because the 210-kDa fragment contained AAA1-4,the expectation was that it might retain much, if not all, ofthe ATPase activity of the complete motor. Surprisingly,ATPase activity was some 6-to 10-fold higher in the 210-kDa fragment than in the complete 380-kDa motor frag-ment. This result provided the Wrst suggestion that the miss-ing portion of the motor domain might containautoinhibitory elements.

The speciWc activity of the 210-kDa fragment showedsome variability, but the highest level of activity was verysimilar to that observed for the complete motor domain in

178 R.B. Vallee, P. Höök / Journal of Structural Biology 156 (2006) 175–181

the presence of microtubules. A simple model to accountfor these data is that microtubule binding relieves anautoinhibitory eVect mediated through elements within theC-terminal half of the motor domain, most likely includingthe stalk.

Another surprising feature of the 210-kDa fragment wasits insensitivity to VO4-mediated photocleavage, a signatureenzymological feature of the dynein protein family. ATPaseactivity of the 210-kDa fragment was also completelyinsensitive to VO4, despite the well-known sensitivity of thedyneins to micromolar concentrations of this ion. ADP-VO4 is generated from hydrolysis of ATP in the presence ofVO4 and mimics the ADP-PO4 transition state. Onceformed, ADP-VO4 remains tightly associated with thedynein active site. The insensitivity of the 210-kDa frag-ment to VO4 suggested that the kinetics of substrate bind-ing might be altered.

This possibility was explored in two ways. First, solventaccessibility of the active sites was assessed by quenching ofdynein-bound 2�-deoxy-mant-ADP with acrylamide.Quenching was substantially increased for the 210-kDafragment, consistent with a more open conformation rela-tive to the 380-kDa motor fragment. Second, the rate ofADP release was examined. This step in the enzymatic cyclehas been found to be rate-limiting for axonemal dyneins(Holzbaur and Johnson, 1989). ADP release for the recom-binant cytoplasmic dynein motor was increased »100-foldfor the 210-kDa fragment relative to the complete motordomain. A similar eVect, though even greater in magnitude(»1000-fold), was produced by exposure of the 380-kDamotor fragment to microtubules. These results suggest thatmicrotubule binding to the stalk relieves inhibition of prod-uct release from the dynein active sites. In this view, there-fore, microtubule binding to the stalk is detected by thedynein ATPase sites as the conformational equivalent ofthe removal of the stalk.

We noted that the dissociation kinetics for 2�-deoxy-mant-ADP from either the 210- or 380-kDa fragments werebiphasic with major and minor components. Each phasewas stimulated to approximately the same extent by micro-tubules. Similarly, each phase was increased in the 210-kDaversus the 380-kDa motor fragment. These data presum-ably reXect major and minor active sites, which are regu-lated proportionately by the stalk. The two phases arelikely to represent activities at AAA1 and AAA3 based onmutational analysis, which revealed a similar apportion-ment of steady state ATP hydrolytic activity betweenAAA1 and AAA3 (Kon et al., 2004).

5. Evidence from proteolytic digestion for control of dynein mechanochemistry by a C-terminal domain

To learn more about the structural organization andintramolecular regulation of the dynein motor domain, wesubjected both the 380- and 210-kDa fragments to con-trolled proteolytic digestion (Höök et al., 2005). Surpris-ingly, the number of discrete fragments that could be

identiWed was very small. Using N-terminal Edman degra-dation and mass spectrometry, we were able to deWne thecleavage sites precisely. Within the 380-kDa motor domain,trypsin cleavage was detected at only three sites (Fig. 1).The Wrst site was within 31 a.a. of the N-terminus of theconstruct, indicating that the engineered N-terminus liesclose to or within a natural interdomain boundary. A com-parable (but nonidentical) tryptic site was detected in thedigest of the 210-kDa fragment. A second tryptic site wasidentiWed at a.a. 2395. This was the only site identiWed in thecentral region of the motor domain, and lies within AAA2.This site is common to both the 380- and 210-kDa con-structs, and identical to a tryptic cleavage site identiWed innative rat cytoplasmic dynein (Mikami et al., 1993), provid-ing additional support for proper folding of both of therecombinant polypeptides. A third site was identiWed 282a.a. from the C-terminus of the 380-kDa construct, justafter AAA6 and within the portion of the motor missing inthe 210-kDa construct. Using the broader speciWcity prote-ases subtilisin and papain, a similar limited pattern of dis-crete fragments was produced from the 380- and 210-kDaconstructs, though the precise sites of proteolytic cleavagewere somewhat diVerent from those identiWed for trypsin.Together, these data suggested a remarkable degree of com-pactness to the dynein motor domain. Furthermore, theproteases cleaved at none of the predicted subdomainboundaries, including the junctions between linker andAAA1, between AAA units, and the N- and C-terminalborders of the stalk. A previous proteolytic analysis of axo-nemal dynein identiWed two tryptic sites within the motordomain, each of which is located within the stalk, one in theglobular microtubule-binding domain and the other closeto the C-terminal boundary of the stalk (Mocz et al., 1991).No evidence for cleavage within AAA2 or toward the C-terminus of the motor domain was obtained in either case.The AAA2 site in cytoplasmic dynein lies within a pre-dicted lysine-rich loop, unique to cytoplasmic dyneins.Therefore, the sensitivity of this site is unlikely to representgreater structural Xexibility in the cytoplasmic dynein AAAring, but rather the presence of an extra protease-sensitiveloop. Size exclusion chromatography of tryptic digests ofthe cytoplasmic dynein motor revealed that the fragmentsproduced by cleavage at this site remained associated innon-denaturing buVer, suggesting that cleavage within theloop represents a relatively innocuous nick in an otherwisehighly condensed motor structure.

The C-terminal cleavage site, in contrast, appears to cor-respond to a bona Wde interdomain boundary, and to deWnea novel structural domain within the dynein motor (Hööket al., 2005). Proteolysis produces a 32-kDa fragment,which is released from the rest of the motor domain asjudged by size exclusion chromatography. Remarkably, thecleavage site is identical to the C-terminal boundary offungi dynein based on sequence alignment (Fig. 2). The C-terminal domain contains no clear functional motifs andshows no clear homology to proteins other than dynein.The region of sequence immediately following AAA6 and

R.B. Vallee, P. Höök / Journal of Structural Biology 156 (2006) 175–181 179

extending to the tryptic site is well conserved in higher andlower eukaryotic dyneins. This region could represent anextension of AAA6, though it could also represent a part ofthe C-terminal domain. The sequence surrounding thecleavage site tends to be high in glycines, serines, and ala-nines, potentially providing structural Xexibility (Fig. 2).Surprisingly, even this proteolytic site could be made resis-tant to proteolysis. Cleavage is observed in the absence ofnucleotide, in the presence of ATP, its nonhydrolyzableanalogue AMPPNP, or ADP. However, cleavage is abol-ished in the presence of ADP-VO4 or ADP-AlF4, both ofwhich behave as transition state analogues. Together, thesedata indicate that the state of ATP hydrolysis aVects theconformation of the C-terminal portion of the motordomain.

Tryptic digestion of the 380-kDa motor domain, con-versely, alters dynein enzymatic activity (Höök et al., 2005),an eVect which may result from cleavage at the C-terminaltryptic site. As for the 210-kDa fragment, the trypsin-treated 380-kDa motor domain is completely insensitive toVO4-mediated photocleavage. In contrast to the 210-kDafragment, the digest exhibits a modest (»40%) decrease insteady state ATPase activity. Which tryptic sites contributeto these eVects is uncertain. We argue that sites 1 and 2(Fig. 1) are not responsible for the eVect on steady-stateATPase activity, because no similar decrease is observed

with cleavage at the same sites within the 210-kDa frag-ment. Cleavage at site 2 leaves the motor domain intact (seeabove Höök et al., 2005), in further support of a minimalenzymatic eVect.

In contrast, cleavage at the C-terminal site does delete aportion of the motor domain, which could aVect enzymaticactivity based on the location of this domain in the circulardynein motor structure. We note that, despite its great dis-tance from AAA1 and AAA3 within the dynein polypeptide(Fig. 1), the C-terminal domain may lie relatively close tothese sites within the folded molecule (Figs. 3 and 4). Weargue that the eVect of nucleotides on the sensitivity of the C-terminal site to tryptic cleavage suggests that the N- and C-termini of the motor domain may physically interact, and doso in a regulated manner. Even the means through which theinteraction aVects proteolysis is suggested in the existingultrastructural analysis. Averaged electron microscopicimages of the dynein motor domain show evidence of a rela-tively tenuous link between two globular subdomains(Samso et al., 1998), which may represent AAA6 and the C-terminal domain. Thus, our proteolytic data appear to implythat the enzymatic state of AAA1 and possibly AAA3 aVectthe conformation of the link between AAA6 and the C-ter-minal domain, thereby limiting access to proteases.

Conversely, an interaction between the C- and N-termi-nal parts of the ring-shaped motor domain may regulate

Fig. 2. Comparison of C-terminal sequences among dynein heavy chains. A 32-kDa region is found in most eukaryotic cytoplasmic and axonemal dyneins.Fungal dyneins lack the region entirely (as in S. pombe), or they retain either a short region up to a position corresponding to the tryptic cleavage site in ratcytoplasmic dynein (arrow), as shown. The junction between the shorter and longer C-terminal segments contains an unusually high number of chargedresidues and residues with small side chains (in bold/red). These residues may confer for Xexibility to this region and permit movement of the C-terminusrelative to the rest of the dynein motor.

180 R.B. Vallee, P. Höök / Journal of Structural Biology 156 (2006) 175–181

ATPase activity, as suggested by the eVects of trypsin onVO4-mediated photocleavage by the 380-kDa motordomain. The steady-state ATPase activity of the latterpreparation was much lower than that of the 210-kDarecombinant motor fragment. This observation is verymuch consistent with the absence of more than one regula-tory element in the 210-kDa polypeptide, e.g., the stalk andC-terminal domains. However, it also implies that thenature of regulation by the C-terminal domain is verydiVerent from that produced by the stalk. Conceivably thestep in the dynein mechanochemical cycle regulated bythe C-terminal domain is diVerent from that aVected by thestalk. Whether this is the case, and why the dynein motordomain should contain multiple regulatory elementsremains to be explored more fully.

Fig. 4. Proposed role of the dynein C-terminal domain in ATPase regula-tion. I: Junction between C-terminal 32-kDa domain and AAA6 is shownas tenuous link accessible to trypsin under basal nucleotide-free, sub-strate-binding, and product-binding conditions. II: Junction between C-terminal 32-kDa domain and AAA6 becomes inaccessible to trypsin in thetransition state (ATP¤), mimicked by ADP-VO4 and ADP-AlF4. C-Termi-nal domain is also deduced to interact more intimately with AAA1 toaccount for the eVect of tryptic removal on ATPase and VO4-mediatedphotocleavage activities. Adapted from Höök et al. (2005).

ATP*

ATP*

I II

Thus, dynein appears to have two diVerent regionsresponsible for regulating enzymatic activity, the stalk andthe newly identiWed C-terminal domain. Microtubule bindingto the stalk produces the same eVect on product release andsteady state ATPase activity as complete absence of thisstructure. Thus, the stalk serves as an autoinhibitory elementthat is inactivated by microtubules. The speciWc role of the C-terminal domain is less clear. Its removal decreases steady-state ATPase activity. Which step in the cross-bridge cycle itregulates remains to be determined. The absence of thisdomain in some organisms indicates that it is not necessaryfor dynein function, though we suspect that it must confer alevel of regulation required in higher eukaryotes.

Acknowledgments

This work was supported by NIH Grant GM47434 toR.B.V. and an AHA fellowship to P.H.

References

Burgess, S.A., Walker, M.L., Sakakibara, H., Knight, P.J., Oiwa, K., 2003.Dynein structure and power stroke. Nature 421, 715–718.

Gee, M.A., Heuser, J.E., Vallee, R.B., 1997. An extended microtubule-bind-ing structure within the dynein motor domain. Nature 390, 636–639.

Gibbons, I.R., Gibbons, B.H., Mocz, G., Asai, D.J., 1991. Multiple nucleo-tide-binding sites in the sequence of dynein � heavy chain. Nature 352,640–643.

Gibbons, I.R., Lee-Eiford, A., Mocz, G., Phillipson, C.A., Tang, W.-J.Y.,Gibbons, B.H., 1987. Photosensitized cleavage of dynein heavy chains:cleavage at the “V1 site” by irradiation at 365 nm in the presence ofATP and vanadate. J. Biol. Chem. 262, 2780–2786.

Goodenough, U.W., Gebhart, B., Mermall, V., Mitchell, D.R., Heuser, J.E.,1987. High-pressure liquid chromatography fractionation of Chla-mydomonas dynein extracts and characterization of inner-arm dyneinsubunits. J. Mol. Biol. 194, 481–494.

Goodenough, U.W., Heuser, J.E., 1984. Structural comparison of puriWeddynein proteins with in situ dynein arms. J. Mol. Biol. 180, 1083–1118.

Fig. 3. Proposed role of dynein stalk in regulating product release from AAA1 and AAA3. I: Unstimulated complete motor domain is shown. ATP hydro-lysis occurs at basal levels, with a greater contribution from AAA1 and a lesser contribution from AAA3. II: Binding of microtubule (not to scale) to stalkstimulates ATP hydrolysis as a result of increased rate of ADP release from AAA1 and AAA3. III: 210-kDa partial motor domain exhibits comparableincreased ATPase activity and ADP release rate as in condition II. These observations suggest that stalk is an autoinhibitory element, the eVects of whichon ATP hydrolysis are eliminated by microtubule binding or stalk removal. Adapted from Höök et al. (2005).

ATPADP

ATPADP

ATPADP

ATPADP ADP

ATP

ATPADP

I II III

R.B. Vallee, P. Höök / Journal of Structural Biology 156 (2006) 175–181 181

Grissom, P.M., Vaisberg, E.A., McIntosh, J.R., 2002. IdentiWcation of aNovel Light Intermediate Chain (D2LIC) for Mammalian Cytoplas-mic Dynein 2. Mol. Biol. Cell 13, 817–829.

Habura, A., Tikhonenko, I., Chisholm, R.L., Koonce, M.P., 1999. Interac-tion mapping of a dynein heavy chain. IdentiWcation of dimerizationand intermediate-chain binding domains. J. Biol. Chem. 274,15447–15453.

Holzbaur, E.L., Johnson, K.A., 1989. ADP release is rate limiting insteady-state turnover by the dynein adenosinetriphosphatase. Bio-chemistry 28, 5577–5585.

Höök, P., Mikami, A., Shafer, B., Chait, B.T., Rosenfeld, S.S., Vallee, R.B.,2005. Long range allosteric control of cytoplasmic dynein ATPaseactivity by the stalk and C-terminal domains. J. Biol. Chem. 280,33045–33054.

Höök, P., Vallee, R. The Dynein Family at a Glance. J. Cell. Sci. in preparation.Kon, T., Mogami, T., Ohkura, R., Nishiura, M., Sutoh, K., 2005. ATP

hydrolysis cycle-dependent tail motions in cytoplasmic dynein. Nat.Struct. Mol. Biol. 12, 513–519.

Kon, T., Nishiura, M., Ohkura, R., Toyoshima, Y.Y., Sutoh, K., 2004. Dis-tinct functions of nucleotide-binding/hydrolysis sites in the four AAAmodules of cytoplasmic dynein. Biochemistry 43, 11266–11274.

Mikami, A., Paschal, B.M., Mazumdar, M., Vallee, R.B., 1993. Molecularcloning of the retrograde transport motor cytoplasmic dynein (MAP1C). Neuron 10, 787–796.

Mikami, A., Tynan, S.H., Hama, T., Luby-Phelps, K., Saito, T., Crandall,J.E., Besharse, J.C., Vallee, R.B., 2002. Molecular structure of cytoplas-mic dynein 2 and its distribution in neuronal and ciliated cells. J. CellSci. 115, 4801–4808.

Mocz, G., Farias, J., Gibbons, I.R., 1991. Proteolytic analysis of domainstructure in the � heavy chain of dynein from sea urchin sperm Xagella.Biochemistry 30, 7225–7230.

Neuwald, A.F., Aravind, L., Spouge, J.L., Koonin, E.V., 1999. AAA+: Aclass of chaperone-like ATPases associated with the assembly, opera-tion, and disassembly of protein complexes. Genome Res. 9, 27–43.

Paschal, B.M., Shpetner, H.S., Vallee, R.B., 1987. MAP 1C is a microtu-bule-activated ATPase which translocates microtubules in vitro andhas dynein-like properties. J. Cell. Biol. 105, 1273–1282.

Paschal, B.M., Vallee, R.B., 1987. Retrograde transport by the microtubuleassociated protein MAP 1C. Nature 330, 181–183.

Pazour, G.J., Dickert, B.L., Witman, G.B., 1999. The DHC1b (DHC2) iso-form of cytoplasmic dynein is required for Xagellar assembly. J. CellBiol. 144, 473–481.

Perrone, C.A., Tritschler, D., Taulman, P., Bower, R., Yoder, B.K., Porter,M.E., 2003. A novel Dynein light intermediate chain colocalizes with

the retrograde motor for intraXagellar transport at sites of axonemeassembly in chlamydomonas and Mammalian cells. Mol. Biol. Cell 14,2041–2056.

Porter, M.E., 1996. Axonemal dyneins: assembly, organization, and regula-tion. Curr. Opin. Cell Biol. 8, 10–17.

Porter, M.E., Bower, R., Knott, J.A., Byrd, P., Dentler, W., 1999. Cytoplas-mic dynein heavy chain 1b is required for Xagellar assembly in Chla-mydomonas. Mol. Biol. Cell 10, 693–712.

Reck-Peterson, S.L., Vale, R.D., 2004. Molecular dissection of theroles of nucleotide binding and hydrolysis in dynein’s AAAdomains in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA101, 1491–1495.

Sakakibara, H., Kojima, H., Sakai, Y., Katayama, E., Oiwa, K., 1999.Inner-arm dynein c of Chlamydomonas Xagella is a single-headed pro-cessive motor. Nature 400, 586–590.

Samso, M., Radermacher, M., Frank, J., Koonce, M.P., 1998. Structuralcharacterization of a dynein motor domain. J. Mol. Biol. 276, 927–937.

Shpetner, H.S., Paschal, B.M., Vallee, R.B., 1988. Characterization of themicrotubule-activated ATPase of brain cytoplasmic dynein (MAP 1C).J. Cell Biol. 107, 1001–1009.

Signor, D., Wedaman, K.P., Orozco, J.T., Dwyer, N.D., Bargmann, C.I.,Rose, L.S., Scholey, J.M., 1999. Role of a class DHC1b dynein in retro-grade transport of IFT motors and IFT raft particles along cilia, butnot dendrites, in chemosensory neurons of living Caenorhabditis ele-gans. J. Cell Biol. 147, 519–530.

Silvanovich, A., Li, M.G., Serr, M., Mische, S., Hays, T.S., 2003. The thirdP-loop domain in cytoplasmic dynein heavy chain is essential fordynein motor function and ATP-sensitive microtubule binding. Mol.Biol. Cell 14, 1355–1365.

Straube, A., Enard, W., Berner, A., Wedlich-Soldner, R., Kahmann, R.,Steinberg, G., 2001. A split motor domain in a cytoplasmic dynein.Embo J. 20, 5091–5100.

Tynan, S.H., Gee, M.A., Vallee, R.B., 2000. Distinct but overlapping siteswithin the cytoplasmic dynein heavy chain for dimerization and forintermediate chain and light intermediate chain binding [In ProcessCitation]. J. Biol. Chem. 275, 32769–32774.

Vale, R.D., 2003. The molecular motor toolbox for intracellular transport.Cell 112, 467–480.

Vallee, R.B., Wall, J.S., Paschal, B.M., Shpetner, H.S., 1988. Microtubuleassociated protein 1C from Brain is a two-headed cytosolic dynein.Nature 332, 561–563.

Vallee, R.B., Williams, J.C., Varma, D., Barnhart, L.E., 2004. Dynein: Anancient motor protein involved in multiple modes of transport. J. Neu-robiol. 58, 189–200.