Kinesin’s light chains inhibit the head- andmicrotubule-binding activity of its tailYao Liang Wong and Sarah E. Rice1

Department of Cell and Molecular Biology, Northwestern University, Chicago, IL 60611

Edited by James A. Spudich, Stanford University School of Medicine, Stanford, CA, and approved May 25, 2010 (received for review April 28, 2010)

Kinesin-1 is a microtubule-based motor comprising two heavychains (KHCs) and two light chains (KLCs). Motor activity is pre-cisely regulated to avoid futile ATP consumption and to ensureproper intracellular localization of kinesin-1 and its cargoes. TheKHC tail inhibits ATPase activity by interacting with the enzymaticKHC heads, and the tail also binds microtubules. Here, we present arole for the KLCs in regulating both the head- and microtubule-binding activities of the kinesin-1 tail. We show that KLCsreduce the affinity of the head-tail interaction over tenfold andconcomitantly repress the tail’s regulatory activity. We also showthat KLCs inhibit tail-microtubule binding by a separate mechan-ism. Inhibition of head-tail binding requires steric and electrostaticfactors. Inhibition of tail-microtubule binding is largely electro-static, pH dependent, and mediated partly by a highly negativelycharged linker region between the KHC-interacting and cargo-binding domains of the KLCs. Our data support a model whereinKLCs promote activation of kinesin-1 for cargo transport by simul-taneously suppressing tail-head and tail-microtubule interactions.KLC-mediated inhibition of tail-microtubule binding may alsoinfluence diffusional movement of kinesin-1 on microtubules, andkinesin-1’s role in microtubule transport/sliding.

regulation ∣ molecular motor ∣ fluorescence anisotropy

Molecular motors (kinesins, myosins, and dyneins) driveactive transport processes within the cell (1–3). Precise con-

trol of each motor’s activity is critical for cell survival. Biochem-ical experiments have determined the detailed mechanisms oftheir isolated, truncated enzymatic domains, but have lagged be-hind cell biology in examining the effects of motor regulators and/or cargo-binding elements. The current picture is that motors areessentially binary, switching between motile (“on”) and regulated(“off”) states, whereas different cargo adapters simply select thecargo that is transported. Cell biological data have made it clearthat this view is an oversimplification, as regulators can toggle aspectrum of different functions and activities for a single motor,depending on cellular context (2, 4, 5). These emerging discov-eries in the kinesin field parallel the development of our under-standing of muscle myosin, which we now know switches betweenmyriad distinct functions as its regulators respond to specific phy-siological conditions (6–8). In this work, we have focused on thekinesin-1 motor as a starting point for biochemically examiningthe complex interplay between kinesin motor activity and regu-latory subunits. Our results yield principles that are conceptuallyapplicable across motor families.

Kinesin-1 is the most well-studied member of the conservedkinesin superfamily. The basic motile and regulatory mechanismsof kinesin-1 are understood (9–14). However, detailed structuraland biochemical studies of the kinesin-1 holoenzyme, comprisingtwo heavy chains (KHCs) and two light chains (KLCs), are morelimited (15–18). Kinesin-1 performs multiple distinct transportfunctions simultaneously within the same cell. In neurons, ittransports vesicular cargoes persistently for long distances alongthe axon (19), positions and anchors mitochondria on microtu-bules (MTs) in response to metabolic demand (20), and activelytransports short MTs for axonal growth and maintenance (21, 22).Distinct cargo adapters mediate each of these kinesin-1 functions.

KLCs are the cargo adapters for neuronal vesicles and the ma-jority of organelles that are transported by kinesin-1, whereas theMiro/Milton complex links kinesin-1 to mitochondria (23–25).The C-terminal kinesin-1 tail can bind ribonucleoprotein cargoes(26) and has a nucleotide-independent MT-binding activity thatmay facilitate kinesin-1’s MT transport/sliding function (27–30).All these distinct apparatuses are located near the critical auto-regulatory region of the KHC tail. Thus, in addition to bindingthe motor to different cargoes, these elements could tailor kine-sin-1 activity for specific transport functions.

Studies on kinesin-1 function and regulation in cells have beenhampered by apparent inconsistencies, which likely arise fromdifferent regulators triggering different dominant activities. Aconserved region in the KHC tail, which we refer to as thehead/MT-binding (HM) site, mediates two distinct functions:(i) binding to the enzymatic KHC heads for inhibition of motoractivity (31) and (ii) binding to MTs (11, 28). When kinesin-1 isautoinhibited, the KHC tails interact directly with the KHC heads(14). The tailHMsitemediates tight binding to thehead, so that anadjacent inhibitory motif can block ADP release (10, 14, 31–33).While existing results agree that the KLCs are also involved in reg-ulation, they appear to be contradictory as to whether the KLCsactivate (16) or inhibit (17, 18) kinesin-1 motor activity.

The tail HM site can also bind MTs independent of the head(28). It has been suggested that MT binding by the tail allowskinesin-1 to slide and organize MT arrays (27, 29, 30). TheHM site may also play a role in nucleotide-independent diffusionof kinesin-1 on MTs. This “energy-saving” mode of attachmenthas been demonstrated in many motors, including kinesin-1(34–36). The affinity of the tail-MT interaction is very high(Kd ¼ 0.4 μM at 200 mM NaCl) (37). This leads to a second ap-parent contradiction in the existing data on kinesin-1 regulation.Despite the high affinity of the tail for both KHC heads and MTs,kinesin-1 is not permanently trapped in an inactive conformationnor perpetually tail-tethered to a MT in a transport-incompetentstate. Negative regulators of the tail must exist, and this couldsuggest the influence of cell-specific factors. For example, theproportion of KHCs associated with KLCs may vary, both withina cell and between cell types (38). Additionally, extrinsic bindingpartners of kinesin-1 have been shown to alter its behavior(23, 39–41). However, no detailed mechanism has been identifiedthat toggles kinesin-1 between its regulated, MT transport/slidingand cargo transport states.

Interestingly, single-molecule motility experiments revealedthat purified kinesin-1 holoenzymes and truncated head dimershad similar processivity, while full-length KHCs were 2.5-fold

Author contributions: Y.L.W. and S.E.R. designed research; Y.L.W. performed research;Y.L.W. and S.E.R. contributed new reagents/analytic tools; Y.L.W. analyzed data; andY.L.W. and S.E.R. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence should be addressed at: Department of Cell and MolecularBiology, Northwestern University, 303 East Chicago Avenue, Ward 8-321, Chicago, IL60611. E-mail: s-rice@northwestern.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1005854107/-/DCSupplemental.

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more processive (32). This suggests that KLCs may intrinsicallyblock MT tethering by the tail, even in the absence of other cel-lular factors. This result, together with their ambiguous functionin motor regulation, prompted us to investigate the effect ofKLCs on kinesin-1 activity and tail-mediated regulation in aminimally reconstituted system.

Here, we show that KLCs are activators of the kinesin-1holoenzyme. By simultaneously suppressing tail-head and tail-MT interactions, they put the motor in a poised state that ismore easily activated for cargo transport. Our results reconcileapparent contradictions regarding KLC function that have beenlong-standing in the field and suggest that kinesin-1 activity isexquisitely balanced, such that mild changes in conditions maydramatically alter motor function. KLC-mediated inhibition oftail-MT binding may also have significant implications for thediffusional movement of the motor, and kinesin-1’s putative rolein MT sliding. It is likely that subunits of other motors exhibit asimilar multipurpose capacity as the KLCs, enabling them toefficiently toggle between distinct functional modes.

Results and DiscussionKinesin-1 Tails Bind Directly to and Inhibit Heads with High Affinity.KLCs have been previously identified as regulators of kinesin-1 activity (17, 18). These authors found that MT binding byKHC/KLC complexes in cell extracts and in live cells was signif-icantly weaker than by KHCs alone. They proposed that KLCsmight enhance inhibition of the kinesin-1 head by the tail. Weexamined this possibility with purified components in vitro, byusing fluorescence anisotropy to directly measure the effect ofthe KLCs on head-tail binding affinity. We used a mutantDrosophila KHC head dimer (Head401; Fig. 1A) with no sur-face-exposed Cys residues. An S195C mutation was introduced,allowing us to site-specifically label Head401 with fluorescein-5-maleimide (Fig. 1B). S195 is close to where the tail binds to thehead (14, 33), and the head-tail interaction results in a readilydetectable anisotropy change.

The tail-interacting surface on the head has an acidic characterwith no positively charged residues (14, 42) (Fig. 1B). Correspond-ingly, a stretch of basic residues in the kinesin-1 tail (aa 927–937)is required for tight binding to the head (31). For our experiments,we used a truncated tail construct (aa 790–975) N-terminallyfused to maltose-binding protein for enhanced solubility (Tail975;Fig. 1A).

We titrated Tail975 into a fixed concentration of labeledHead401 S195C (Fig. 2A, squares). The resulting binding curveshows that KHC head-tail affinity is extremely high, with Kd ¼0.041� 0.004 μM (all concentrations reported as monomers).To functionally correlate this binding data to the activity of theheads, we also performed MT-stimulated ATPase assays (Fig. 2B,squares). The calculated Kd from the functional assay was0.076� 0.003 μM, approximately twofold higher than the valueobtained by fluorescence anisotropy. The results of this functionalassay are reasonably consistent with prior work demonstratingsimilar functional inhibition of kinesin-1 heads by shorter tail con-structs (10, 31). The difference in the affinity measured by aniso-tropy vs. MT-stimulated ATPase may reflect some nonspecificbinding in the anisotropy assay due to the hydrophobicity of thefluorescent probe, which is fairly common. Alternatively, it couldsuggest that the head-tail interaction is more complex than pre-sumed, and that there are events downstreamof initial contact thatinfluence the tail’s inhibitory activity. Importantly, we note thatthe difference in affinities measured by either technique is con-stant in the following experiments, allowing us to assess the effectof KLCs on head-tail binding.

KLCs Inhibit Kinesin-1 Head-Tail Binding. We first confirmed that allour KLC constructs form highly stable complexes with Tail975(Fig. S1), as has been shown for native KHCs (43). Next, we mea-

sured head-tail affinity in the presence of full-length KLCs (KLC-FL; Fig. 1A). At saturating KLC-FL, the Kd of the head-tail in-teraction was decreased 11-fold to 0.440� 0.035 μM (Fig. 2A,diamonds). In the functional ATPase assay, KLCs produced aconcomitant decrease in tail-mediated inhibition of kinesin-1ATPase activity (Fig. 2B, diamonds; Kd ¼ 0.711� 0.050 μM),presumably by reducing the propensity for kinesin-1 to be inits regulated state.

KLCs could reduce the affinity of theKHChead-tail interactionby either electrostatic or steric interactions, or both. The KLCprimary sequence has an overall negative charge (pI ¼ 5.9). Inparticular, the KHC-binding heptad repeats of the KLCs havenegative charges located in close proximity to the tail, where they

Fig. 1. Constructs used in this study. (A) Tail (blue), KLC (red/light gray), andhead (dark gray) constructs are shown beside a cartoon of kinesin-1. Coiled-coil regions in each construct are striped. Residues are numbered accordingto the Drosophila kinesin-1 sequence. Tail975 is a KHC tail N terminally fusedto maltose-binding protein and is colored blue to indicate its basic nature.KLC-FL is the full-length KLC protein. KLCΔTPR is truncated immediatelyN-terminal to the cargo-binding TPR domains, whereas KLC-CC consists ofonly the coiled-coil region of the KLCs. Parts of the KLCs are colored redto indicate acidic regions. Head401 is a KHC head dimer with all surface-exposed Cys residues removed. (B) Crystal structure of the KHC head (PDBID code 1mkj) (42) with S195 indicated in green space-fill representation.Acidic residues on the head’s tail-interacting face, particularly helix α3, arecolored red. There are no basic residues on this face of the motor domain.(C) Figure legend for component depictions in subsequent figures.

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may electrostatically interfere with the tail binding to the nega-tively charged face of the KHC head. The tetratricopeptide repeat(TPR) domain of the KLCs could also sterically block head-tailinteractions.

To distinguish between these two potential means of inhibition,we generated a KLCΔTPR construct (Fig. 1A) and testedwhether it could inhibit head-tail binding. KLCΔTPR consistsof the KHC-interacting domain of the KLCs, plus a highly acidic30-aa disordered region (aa 149–179, pI ¼ 4.1; Fig. S2A) situatedbetween the KHC-binding and cargo-binding domains. Thislinker region is positioned near the tail HM site, assuming thepreceding regions of the KLC are coiled coil. We observed thatKLCΔTPR decreased the affinity of the head-tail interaction ap-proximately 3.5-fold (Fig. 2A, circles; Kd ¼ 0.145� 0.016 μM).In the ATPase assay, KLCΔTPR also exhibited an intermediateeffect (Fig. 2B, circles; Kd ¼ 0.341� 0.021μM) between tails

alone and tailsþ KLC-FL. Thus, whereas steric factors are im-portant for inhibition of tail binding to heads, there is a significantnonsteric component that could be accounted for by an electro-static clash mechanism.

Two past results, together with our data, suggest a possible me-chanism for the KLC-mediated effects on regulation. Hackney etal. showed that head dimerization is required for high affinityhead-tail binding, and tail-mediated inhibition of motor activityoccurs at a stoichiometry of 1 tail∶2 heads (31). This indicatesthat the tail may make stabilizing interactions by nestling in be-tween the two heads. Based on in vivo FRET measurements, Caiet al. reported that the KLC TPR domains separate the KHCheads (18). In this case, the TPRs would interfere with the spe-cific orientation of the dimerized heads required for tail bindingand regulation of the KHC heads.

Cai et al. also reported that a construct similar to KLCΔTPRincreased the in vivo FRET efficiency between fluorescent tagslocated on KHC heads and tails (i.e., heads and tails are closertogether in the presence of this KLC construct). This appears tocontradict our results, but importantly, the heads and tails couldbe in close proximity even if KLCs inhibit tail-mediated regula-tion by the mechanism described above. It is also likely that othercellular factors such as extrinsic binding partners could furthermodify the behavior of kinesin-1 in cells.

KLCs Inhibit Tail-MT Binding. As previously mentioned, the posi-tively charged residues in the tail HM site are important for bothhead and MT binding (31). We recently reported that C-termin-ally truncated human kinesin-1 tail constructs interact electrosta-tically with MTs (37). This mode of binding is common among avariety of MT-associated proteins (44), including the tail ofkinesin-14, which drives directional sliding of antiparallel MTs(45, 46). Given the highly basic nature of the tail HM site in bothhuman and Drosophila kinesin-1 (pI ¼ 11.3 for Drosophila aa910–950), we considered whether our full-length tail domainalso bound to MTs via electrostatic interactions with the acidicC terminus of tubulin.

We found that Tail975 cosedimented with MTs when mixtureswere centrifuged (Fig. S3A). At 3 μM MTs, 85% of Tail975 wasfound in the pellet (Fig. S3B). Subtilisin digestion of MTs to re-move the negatively charged C terminus of tubulin reduced theamount of Tail975 copelleting with MTs to 33% (Fig. S3A and B).The Kd of the tail-MT interaction increased 12-fold (0.5 μM to6 μM) after subtilisin treatment. These results show that MTbinding by full-length kinesin-1 tails is mediated largely by elec-trostatic interactions with the tubulin C terminus.

We then assayed tail-MT binding in the presence of KLC-FL(Fig. 3A). We reasoned that if KLC-mediated inhibition of thetail-head interaction involves a charge clash, the KLCs shouldlikewise inhibit the tail’s electrostatic interaction with MTs.KLC-FL by itself did not bind to MTs, and it inhibited thetail-MT interaction in a concentration-dependent manner(Fig. 3B). The percentage of Tail975 cosedimenting with MTs de-creased from 89% at 0 μM KLC-FL to 24% at 2.5 μM KLC-FL.Importantly, inhibition was approximately saturating at the 1∶1physiological ratio of KHC:KLC, indicating a specific effect(27% of tail in the pellet at 2 μM Tail975 and 2 μM KLC-FL).

Inhibition of Tail-MT Binding Is Mediated by the KLC N Terminus and IspH Dependent.We tested whether KLC-mediated inhibition of thetail-MT interaction was due to steric hindrance from the KLCTPR domains, electrostatic clash, or both. Here, we usedKLCΔTPR as well as a slightly shorter construct lacking the30-aa acidic linker region (KLC-CC; Fig. 1A). At a saturatingratio of KLC:tail, both constructs inhibited tail-MT binding(Fig. 4A), with KLCΔTPR being more effective (17% of Tail975in pellet compared to 43% for KLC-CC; Fig. 4B). These data in-dicate that the TPR domains are not required for inhibition of

Fig. 2. KLCs inhibit tail binding to the heads. (A) Fluorescence anisotropybinding curves of Tail975 to 0.01 μM Head401 in the absence of KLCs(n ¼ 13; squares), and in the presence of KLC-FL (n ¼ 8; diamonds) orKLCΔTPR (n ¼ 9; circles). Data points are mean� SD. (Inset) A Scatchard plotof the data and corresponding fits, along with Kd values calculated by non-linear regression. (B) Head401 ATPase activity in the presence of 2 μM MTs isprogressively inhibited by increasing Tail975 (squares), with a maximumextent of inhibition of 76%. In the presence of KLC-FL (diamonds) orKLCΔTPR (circles), the Kd of the functional inhibition of heads by tails isincreased. Data points are normalized mean� SD (n ¼ 5–15 for each point).

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MT binding. Furthermore, the observation that KLCΔTPR(pI ¼ 4.8) has a higher inhibitory activity than KLC-CC(pI ¼ 5.1) and KLC-FL (pI ¼ 5.9, 24% of tail bound to MTs;Fig. 3B) is consistent with electrostatic clash being the dominantinhibitory mechanism. Even though KLC-FL containing the po-sitively charged TPR domains (pI ¼ 8.5) is more basic overallthan KLC-CC, it was a more potent inhibitor of tail-MT binding.This argues that the acidic linker region is an important factor forinhibition.

Finally, we tested whether inhibition was pH dependent, asexpected for a finely tuned electrostatic system. For these studies,we used KLCΔTPR because the TPR domains in KLC-FL arehighly aggregation-prone below pH 7 in vitro. KLC-mediatedinhibition of tail-MT binding was evident at a physiological pHof 7.4, as shown above. MT binding by the tail alone was not mea-surably affected by a shift to acidic conditions (pH 6.6; Fig. 4 Cand D). However, KLCs became significantly less effective inhi-bitors of the tail-MT interaction. The percentage of Tail975 thatcosedimented with MTs more than doubled, from 17% at pH 7.4to 38% at pH 6.6 (Fig. 4 C and D). This increase in tail-MT bind-ing was not due to KLCs dissociating from Tail975, as an equalproportion of KLCΔTPR shifted into the MT pellet (Fig. 4C),indicating a tail-complexed population.

In the past, some KLC and tail constructs have displayederratic behavior in vitro, possibly due to misfolding, aggregation,or proteolysis issues. Indeed, KHC tails are prone to proteolysis(15), and isolated KLC TPR domains have limited solubility (17).At the near-physiological ionic strength used in our assays, ourconstructs are intact and fully soluble, yielding self-consistentresults (see also SI Results and Discussion).

KLCs Modulate the Affinity of Kinesin-1 Tails for Heads and MTs. Ourobserved KLC-mediated inhibition of head-tail binding is consis-tent with past observations that the equilibrium point of thekinesin-1 holoenzyme (containing KLCs) transition betweencompact (regulated) and extended (active) conformations occursat a lower ionic strength than KHCs alone (16), indicating thatKLCs destabilize the regulated state. In that study, Hackney et al.also observed a pH dependence in the transition: At any given saltconcentration, kinesin-1 was more likely to be in its compact,regulated state at pH 6.3 compared to pH 8.3. This result hasbeen a puzzling contradiction to data obtained by Verhey et al.indicating that the kinesin-1 holoenzyme binds MTs more weaklyand thus should be more regulated at high pH (pH 7.4) than atlow pH (pH 6.6) (17). Our work resolves this incongruity by de-monstrating that while KLCs repress the autoinhibitory head-tailinteraction, they also repress the tail-MT interaction more atphysiological pH than at acidic pH.

Furthermore, this pH dependence suggests that KLC-mediated inhibition may be natively modulated by mechanismsthat could include but are not limited to phosphorylation or otherposttranslational modifications, adapter protein binding, or proxi-mity to localized acidic environments (e.g., around lysosomes orsites of Hþ flux). Cell-specific differences in these mechanismsmay lead to different apparent localization of kinesin-1 in vivo(17, 28, 38). While the in vitro measurements of kinesin-1 foldingby Hackney et al. (16) and our current data still appear to contra-dict the in vivo FRETresults fromCai et al. (18), all of these resultsindicate that because kinesin-1 function is balanced betweendifferent activities, its behavior may be significantly altered inthe cellular milieu relative to isolated in vitro conditions.

Fig. 4. Inhibition of tail-MT binding does not require the KLC TPR domainsand is pH dependent. Protein concentrations were set at 2-μMTail975 � 2.5-μM KLCΔTPR or KLC-CC. (A) KLCΔTPR and KLC-CC inhibitTail975 binding toMTs. (B) The differences in amount of pelleted tail betweenTail975 alone (n ¼ 13), Tail975þ KLCΔTPR (n ¼ 7), and Tail975þ KLC-CC(n ¼ 5) were all highly significant (*, p < 0.001). Estimated tail-MT Kd valueswere >14 μM in the presence of KLCΔTPR and 4 μM in the presence ofKLC-CC. (C)MT-bindingassay performedatpH6.6 andpH7.4. (D) Tail975 alonebinds equally well to MTs at pH 6.6 or pH 7.4. However, KLC-mediatedinhibition of the tail-MT interaction is significantly reduced at pH 6.6, withthe Kd for tail-MT binding in the presence of KLCΔTPR decreasing to approxi-mately 5 μM (*, p < 0.001, n ¼ 5).

Fig. 3. KLCs inhibit tail binding to MTs. (A) KLC-FL did not interact with MTsby itself, and it inhibited the tail-MT interaction in a concentration-depen-dent manner. Tail975 concentration was fixed at 2 μM. (B) Increasing theamount of KLC-FL resulted in a linear decrease in the percentage of pelletedTail975 (n ¼ 6–13). Maximum inhibition was observed at 2-μM KLC-FL.Beyond the 1∶1 ratio of KLC-FL:Tail975, the amount of pelleted tail didnot decrease significantly (p > 0.05). At saturating KLC-FL levels, the Kd

for tail-MT binding was approximately 9 μM.

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In cells, RanBP2 and the fasciculation and elongation ζ 1(FEZ1) protein are likely candidates for extrinsic modulatorsof KHC tail behavior (40, 41). In a two-hybrid screen, FEZ1 bind-ing to kinesin-1 was abolished by mutations in the HM site of theKHC tail (40). Because of the location of the interaction, weexpected FEZ1 to be a direct extrinsic regulator of the HM site.To test whether this was the case, we performed tail-MT bindingassays as above, in the presence of FEZ1. Because we wereunable to express Unc-76 (the Drosophila structural homolog),we used human FEZ1 (38% sequence similarity to Unc-76)for these assays, and the results are interpreted purely qualita-tively. We observed an interaction between Tail975 and FEZ1that was not inhibited by KLCs, consistent with previous results(Fig. S4A) (40). Under saturating levels of FEZ1, we observed alarge decrease in Tail975 pelleting with MTs (Fig. S4 B and C).These results indicate that the intrinsic KLCs, as well as extrinsicbinding partners, target the tail HM site. Therefore, our data es-tablish the tail HM site as a regulatory focal point. The RanBP2protein is also likely to be another extrinsic direct regulator of theHM site (47).

KLCs Fine-Tune Kinesin-1’s Response to Regulatory Inputs. In sum-mary, we have used purified components to demonstrate thatthe KLCs regulate both the head- and MT-binding activity ofthe kinesin-1 tail, which is itself a regulator of KHC activity. In-hibition of both these interactions is likely to involve an electro-static clash between the negatively charged linker region betweenaa 149–179 of the KLCs and the KHC head or MT. Steric effectsfrom the KLC TPR domains play a significant role in KLC-mediated inhibition of head-tail binding. In contrast, KLC-mediated inhibition of the tail-MT interaction is largely electro-static. These distinct KLC-mediated inhibition mechanisms makeintuitive sense when considered retrospectively, as the tail likelybinds in a cleft formed by the dimerized globular head domains,and the presence of steric obstructions would prevent access tothis specific site. In contrast, the MT lattice presents a convexsurface with multiple binding sites, where steric hindrance wouldbe less effective.

It is intriguing that the sequence of the tail HM site is highlyconserved in species expressing KLCs (Fig. S2B). Conversely, or-ganisms that express KHCs without associated KLCs exhibit adifferent motif even though the nearby regulatory region has highsequence identity across all species. These KLC-less KHCs mayhave a specific mechanism of inhibition based on their distinctsequence (48, 49).

We propose a model for kinesin-1 regulation, in the context ofKLC-mediated inhibition of its tail (Fig. 5). By reducing tail-headaffinity, KLCs may allow regulated kinesin-1 to be more easilyengaged for transport. By blocking tail-MT binding, KLCs

circumvent states in which the tail may be unproductively at-tached to MTs instead of cargo. This also prevents kinesin-1 fromsliding MTs unless KLC repression is abrogated. Consistent withthis idea, KLC null mutants exhibit normal MTmovement duringkinesin-1-driven cytoplasmic streaming (50). However, we notethat there is currently insufficient data to propose a specificmodel for kinesin-1 tail-mediated MTsliding, and our results onlydemonstrate a tight interaction between tails and MTs that is in-hibited by KLCs. The intrinsic KLCs and extrinsic binding part-ners like FEZ1 may toggle kinesin-1 between different motileactivities by regulating the tail HM site.

In conclusion, by modulating the tail’s affinity for MTs and theKHC head, the KLCs serve as an intrinsic general regulator of ki-nesin-1 activity. The data presented here provide insight into howNature has engineered the intersubunit interactions within kine-sin-1, balancing its various motor functions and allowing high sen-sitivity toward external inputs.We anticipate that these results willopen avenues for studying how regulators of kinesin-1 and otherkinesin family members engage the motor for distinct activities.

Materials and MethodsCloning and Purification of Constructs. KLC constructs were cloned from cDNAvector GH14842 (Drosophila Genomics Resource Center). Tail aa 790–975were cloned from a full-length Drosophila KHC construct (D. Hackney) andligated to the 3′ end of a maltose-binding protein sequence (W. Anderson).All constructs were C-terminally 6xHis-tagged in pET-17b vectors (Novagen).Head401 was received from N. Guydosh and S. Block. An S195C mutationwas introduced by Quikchange mutagenesis (Stratagene). Proteins were ex-pressed in Escherichia coli BL21(DE3)RP cells with IPTG induction and purifiedon Ni-NTA agarose (Qiagen) using standard protocols (33). Proteins wereeluted in 25 mM Hepes, 300 mM KCl, 300 mM imidazole, 2 mM MgCl2,1 mM EGTA, 0.02% Tween-20, 5% sucrose, 10 mM β-mercaptoethanol,and 20 μM ATP, pH 7.4. Pooled eluate was snap-frozen with an additional15% sucrose (wt∕vol) and stored in liquid nitrogen. Thawed proteins weresoluble and stable for at least 36 h at 4 °C.

Labeling of Head401. Head401 S195C was dialyzed into 25mMHepes, 250 mMKCl, 50 mM imidazole, 1 mM MgCl2, 1 mM EGTA, 0.02% Tween-20, 5%sucrose, 0.2 mM Tris[2-carboxyethyl]phosphine HCl, and 20 μM ATP, pH7.2. Protein concentration was measured using a Bradford protein assay(Thermo Fisher Scientific). A fourfoldmolar excess of fluorescein-5-maleimide(Invitrogen) was mixed with the protein and allowed to react for 16 h at 4 °Cbefore quenching with 25 mM β-mercaptoethanol. Unconjugated dye wasremoved by exchanging into fresh buffer through Amicon Ultracel-50 Kcentrifugal filter devices (Millipore).

Fluorescence Anisotropy. Proteins were dialyzed into assay buffer (25 mMHepes, 100 mM KCl, 20 mM imidazole, 1 mM MgCl2, 1 mM EGTA, 0.02%Tween-20, 5% sucrose, 10 mM β-mercaptoethanol, and 20 μM ATP, pH7.4). Varying amounts of Tail975 were mixed with 0.01 μM labeled Head401S195C� 8 μM KLCs. Reactions were incubated at room temperature for5 min. Anisotropy readings were measured on a Safire II fluorescence micro-

Fig. 5. A model for KLC-mediated regulation of the kinesin-1 tail. Kinesin-1 is colored as in Fig. 1. Without KLCs (Left), kinesin-1 would be either regulated insolution or bound to MTs with the tail tethered. Due to the high affinity of tails for heads/MTs, the motor cannot access its cargo transport-competent state. Inthe presence of KLCs (Right), tail-head and tail-MT interactions are inhibited. Strong inhibition of tail-MT binding means that the regulated conformation ofkinesin-1 becomes the predominant form, but tail-head affinity is also reduced such that the motor is in a poised state that can be easily activated for cargotransport.

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plate reader (Tecan US). Instrument settings were as follows: λex ¼ 470 nm;λem ¼ 525 nm; G-factor correction ¼ 1.1113; Reads per well ¼ 10.

ATPase Measurements. Proteins were dialyzed into assay buffer and ATPaserates were determined using a coupled-enzyme assay (31). The reaction mix-ture contained assay buffer with 1 mM ATP, 2 mM phosphoenolpyruvate,0.26 mM NADH, 3.8 μg∕mL pyruvate kinase, 4.1 μg∕mL lactate dehydrogen-ase, 10 μM taxol, 2 μM MTs, and 0.1 μM Head401� 6 μM KLC. Tail concentra-tions were varied as indicated.

MT-Binding assays. Proteins were dialyzed into 25 mM Pipes (pH 6.6) or Hepes(pH 7.4), 110 mM KOAc, 20 mM imidazole, 1 mM MgCl2, 1 mM EGTA, 0.02%Tween-20, 5% sucrose, and 10 mM β-mercaptoethanol and centrifuged at417;000 × g, 10 min, 4 °C to remove any aggregates. Then 3-μM MTs and0.1-mM taxol were mixed with proteins at the indicated concentrations, in

a final volume of 0.1 mL. Reactions were incubated at room temperaturefor 10 min. MTs and MT-bound proteins were pelleted by centrifugationat 16;000 × g, 25 min. Supernatants and pellets were separated and runon SDS-PAGE. Quantitation was performed in ImageJ. Reported percentagesare given as mean� SD. Student’s t test was used for statistical comparisonbetween conditions. Kd values were calculated from fits to one-site bindingcurves.

ACKNOWLEDGMENTS. We thank N. Guydosh and S. Block for the Head401construct, D. Hackney for the full-length Drosophila kinesin-1 construct,W. Anderson for the maltose-binding protein construct, J. Kobarg for theFEZ1 construct, K. Dietrich and R. Vassar for supplies, D. Freymann for theuse of his equipment, and Park Packing Company for providing materialfor tubulin purification. Y.L.W. and S.E.R. are supported by National Institutesof Health Grant GM072656.

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