Cell, Vol. 96, 69–78, January 8, 1999, Copyright 1999 by Cell PressKin I Kinesins AreMicrotubule-Destabilizing EnzymesErickson and O’Brien, 1992; Desai and Mitchison, 1997).Polymerizing MT ends are thoughttomaintaina stabiliz-ing “cap” of GTP/GDP.Pi-tubulin, the loss of which re-Arshad Desai,*§Suzie Verma,†Timothy J. Mitchison,‡and Claire E. Walczak†k*Department of Biochemistry and Biophysicssults in exposure of an unstable GDP-tubulin core and†Department of Cellular and Molecular Pharmacologyrapid depolymerization. Thus, tubulin has a built-in lat-University of Californiaticedestabilizationmechanismdrivenby GTPhydrolysisSan Francisco, California 94143on b-tubulin. Kinetically, hydrolysis and phosphate re-‡Department of Cell Biologylease result in a GDP-tubulin lattice that has a .1000-Harvard Medical Schoolfold higher subunit off rate from an MT end than GTP-Boston, Massachusetts 02115tubulin (Walker et al., 1988). Structurally, GDP-tubulinprotofilaments are thought to prefer a conformation withincreased outward curvature relative to GTP-tubulinSummaryprotofilaments (Melki et al., 1989; Mandelkow et al.,1991; Mu¨ller-Reichert et al., 1998). In the lattice of aUsing in vitro assays with purified proteins, we showpolymerizing MT, GDP-tubulin protofilaments are con-that XKCM1 and XKIF2, two distinct members of thestrained to being straight, presumably by lattice inter-internal catalytic domain (Kin I) kinesin subfamily, cat-actions, but during depolymerization they can relax intoalyticallydestabilizemicrotubules using anovelmech-the preferred curved conformation. The free energy re-anism. Both XKCM1 and XKIF2 influence microtubuleleased during this relaxation is thought to drive thestability by targeting directly to microtubule endsrapid depolymerization phase of dynamic instability.where they induce a destabilizing conformationalConsistent with these ideas, tubulin polymerized withchange. ATP hydrolysis recycles XKCM1/XKIF2 forGMPCPP, a slowly hydrolyzable GTP analog, forms sta-ble MTs that do not undergodynamic instability(Hymanmultiple rounds of action by dissociating a XKCM1/et al., 1992; Caplow et al., 1994). Taxol, a drug isolatedXKIF2–tubulin dimer complex released upon microtu-from the bark of the yew tree that binds tubulin, alsobule depolymerization. These results establish Kin Istabilizes MTs by suppressing dynamic instability (Hor-kinesinsasmicrotubule-destabilizingenzymes,distin-witz, 1994).guish them mechanistically from kinesin superfamilyMT polymerization dynamics are fundamentally im-members that use ATPhydrolysis to translocatealongportanttotheintracellularfunctionsoftheMTcytoskele-microtubules, and have important implications for theton, as best illustrated by analysis of chromosomeregulation of microtubule dynamics and for the intra-movement (Inoue´and Salmon, 1995). In vivo, the intrin-cellular functions and evolution of the kinesin super-sic dynamic instability of tubulin is extensively regulatedfamily.(Cassimeris, 1993; McNally, 1996; Desai and Mitchison,1997). MTs in vivo turn over much more rapidly thanIntroductionMTs assembled from pure tubulin in vitro, in large partbecause of an increase in the frequency of transitionsMicrotubules (MTs) are noncovalent polar polymers offrom the polymerization phase to the depolymerizationab-tubulinheterodimersfound in alleukaryoticcells thatphase, called the frequency of catastrophe (Belmont etplayanessentialroleincelldivision,cytoplasmicorgani-al., 1990; Verde et al., 1992). This finding is consistentzation, generation and maintenance of cell polarity, andwith modeling studies showing that regulation of catas-many types of cell movements. MTs are inherently dy-trophe frequency is anextremely efficient wayto rapidlynamic polymers that transduce energy derived from nu-modulate MT dynamics (Verde et al., 1992). The ob-cleotide hydrolysis into polymer dynamics. In addition,served high frequency of catastrophe has been sus-the surface of the MT polymer serves as a track onpected to result from the action of cellular proteins thatwhich motor proteins transport cargoes throughout thedestabilizethestabilizing caps atpolymerizingMT ends.Inadditiontobeingdynamicpolymers,MTsalsoservecell. These two distinct facets of the MT polymer areas tracks for motor proteins. Conventional kinesin, thecentral to the many biological functions of the MT cy-founding member of the kinesin superfamily, was identi-toskeleton.fied on the basis of its ability to use energy derived fromMTs are 25 nm diameter, 12–15 protofilament poly-ATP hydrolysis to translocate along the MT lattice (Valemers that utilize polymerization-induced GTP hydrolysisetal.,1985).Inthelastdecade, z100 eukaryotic proteinson b-tubulin to generate dynamic instability—a behaviorhave been identified that contain a domain homologouswhere polymerizing and depolymerizing MTs coexist into the z300 amino acid catalytic ATPase domain ofthe same population, infrequently interconverting be-conventional kinesin (Vale and Fletterick, 1997; Hiro-tween these two states (Mitchison and Kirschner, 1984;kawa et al., 1998). Based solely on sequence alignmentsof their catalytic domains, the majority of kinesins have§To whom correspondence should be addressed at the followingbeen classified into eight subfamilies with a minoritypresent address: European Molecular Biology Laboratory, Meyer-being “orphans” (Vale and Fletterick, 1997). These sub-hofstrasse 1, D-69117 Heidelberg, Germany (e-mail: arshad.desai@family categorizations are supported to varying extentsembl-heidelberg.de).by location of the catalytic domain within the polypep-kPresent address: Indiana University, Medical Sciences, Blooming-ton, Indiana 47405.tide chain of the protein, by analysis of the quaternaryCell70structures of the native proteins, and by the similarityof the biological processes in which members of a sub-family derived from different organisms are implicated.Approximately 20% of identified kinesins have been re-ported to have motor activity in in vitro motility assays.Among the kinesins characterized in vitro is at leastone member of each of the eight subfamilies, stronglysupporting the assertion that kinesins are mechano-chemical ATPases that translocate along the MT lattice.Previously, we described a kinesin, XKCM1, that isinvolved in regulating MT dynamics in frog egg extracts(Walczak et al., 1996). Depletion of XKCM1 resulted ina dramatic increase in MT polymerization,