Isolation of a 45-kDa Fragment from the Kinesin Heavy Chain with

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 264, No. 1, Issue of January 5, pp. 589-595,1989 Printed in U.S.A.

Isolation of a 45-kDa Fragment from the Kinesin Heavy Chain with Enhanced ATPase and Microtubule-binding Activities*

(Received for publication, July 25, 1988)

Sergei A. KuznetsovS, Yevgeny A. Vaisbergs, Stephen W. Rothwellll, Douglas B. Murphyll**, and Vladimir I. GelfandSS From the $Department of Molecular Biology, Biology Faculty and the $$4. N . Belozersky Laboratory of Molecular Biology and Bioorganic Chemistry, Moscow State University, Moscow 119899 and the §Institute of Protein Research, Academy of Sciences of the Union of Soviet Socialist Republics, Pushchino, Moscow Region 142292, Union of Soviet Socialist Republics, the VDepartment of Hematology, Walter Reed Institute for Research, Washington, D. C. 20307-5100, and the 11 Department of Cell Biology and Anatomy, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Kinesin is a microtubule-activated, mechanochemical ATPase capable of moving particles along microtubules and making microtubules glide along a solid substrate. In this study we used limited proteolysis to study the structure of bovine brain kinesin, a hetero- tetramer composed of two heavy (120-kDa) and two light (62-kDa) chains. a-chymotrypsin, trypsin, and subtilisin all produced a protease-resistant 45-kDa fragment from the kinesin heavy chain. As isolated by gel-filtration chromatography, this fragment contains both the microtubule-binding site and the ATP catalytic site of the molecule. Proteolytic cleavage stimulated microtubule-dependent Mg+-ATPase activity 4- to &fold up to 75-120 pmol ATP/min/mg. Cleavage also increased the affinity of the fragment for microtubules at least 10-fold. Since the purified fragment does not support the gliding of flagellar axonemes, we propose that cleavage of the heavy chain uncouples ATPase activity from its translocator activity, which may require other parts of the molecule.

Mammalian nerve cells and many other eukaryotic cells contain at least two microtubule-dependent translocator proteins. These include kinesin (l), which moves particles along microtubules in an anterograde direction from their minus to plus ends (2), and cytoplasmic dynein or MAP-lC, which promotes retrograde movements in the opposite direction (3, 4). Kinesin moves microtubules on glass surfaces and supports microtubule-associated movements of carboxylated beads i n vitro (5 , 6). Dynein is thought to be the retrograde motor for vesicle transport, but so far it is only known to support microtubule gliding (4), although other high molecular weight ATPases possibly related to dynein have been shown to bind or move beads and organelles on microtubules in vitro (7-10). Both kinesin and cytoplasmic dynein are microtubule-activated ATPases (3 , l l ) and are complex, multisubunit proteins.

Bovine brain kinesin is a heterotetrameric molecule composed of two heavy (120-kDa) and two light (62-kDa) chains (12, 13) and is known to hydrolyze ATP, bind microtubules to other substrates, and function in motility i n vitro. However,

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** Participant in the program for scientific exchanges between the National Academy of Sciences of the United States and the Academy of Sciences of the Union of Soviet Socialist Republics; supported in part by Grant GM33171 from the National Institutes of Health.

little is known about the physical relationship of these sites to each other or their distribution on the protein. The relationship of kinesin ATPase to dynein and myosin ATPases is also incompletely understood. As a logical first step in studying the structure of kinesin, we used partial proteolysis to generate fragments containing defined domains of the molecule as this approach proved useful in studying the struc- tures and mechanisms of other complex ATPases such as dynein and myosin.

In this paper we show that a-chymotrypsin, trypsin, and subtilisin produce a relatively stable 45-kDa fragment from the heavy chain of kinesin, which contains both ATPase and microtubule-binding properties but which itself is incapable of translocating microtubule axonemes on a glass surface. We propose that proteolysis removes essential regulatory domains of the molecule and uncouples ATP hydrolysis and translocator activities, resulting in a fragment with increased ATPase and microtubule-binding activities.

MATERIALS AND METHODS

Isolation of Microtubules and Kinesin-Microtubules and tubulin were purified from bovine brain by two cycles of polymerization- depolymerization (14) in buffer A (50 mM imidazole-HC1, pH 6.7, 0.5 mM MgC12, 0.1 mM EDTA, and 1 mM P-mercaptoethanol) supplemented with 50 mM KC1 and 4 M glycerol. Tubulin was purified by chromatography on phosphocellulose P-11 (Whatman, Maidstone Kent, United Kingdom) (15) in buffer A containing 50 mM KC1 and 1 mM EGTA.’ Bovine brain kinesin was purified according to Kuz- netsov and Gelfand (11) using Sephacryl S-300 for the gel filtration step and DEAE-cellulose to concentrate the enzyme. These two steps were performed in the absence of PMSF.

ATPase Assays-ATPase activity was measured at 37 “C in buffer B (50 mM imidazole-HC1 pH 6.7, 3 mM MgC12, 0.1 mM EDTA, 1 mM EGTA, 1 mM P-mercaptoethanol), supplemented with 2 mM ATP, unless otherwise indicated. The K, for ATP was measured in buffer B supplemented with an ATP-regenerating system (2 mM phos- pho(eno1)pyruvate and 1 unit/ml of pyruvate kinase) and various concentrations of ATP. The ATP-regenerating system had no effect on the ATPase activity of intact kinesin. ATPase activity was measured using the colorimetric assay of Panusz et al. (16) for inorganic phosphate.

Proteolytic Digestion and Purification-Kinesin was digested with proteases either in buffer A with 0.15 M KC1 (for purifying peptides from proteolytic digests) or in buffer B (for studies of microtubule binding and ATPase activities in unfractionated digests). Proteolysis was arrested with 2 mM PMSF. To purify peptides, 0.5-ml aliquots of the digest (0.4-0.6 mg/ml) were fractionated at a rate of 0.5 ml/

The abbreviations used are: EGTA, [ethylenebis(oxethyl- enenitri1o)ltetraacetic acid; AMP-PNP, adenylylimidodiphosphate; PMSF, phenylmethylsulfonyl fluoride; PPPi, inorganic tripolyphos- phate; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

589

590 ATPase-containing Fragment of Kinesin min on a fast protein liquid chromatography HR 10/30 column (Pharmacia Fine Chemicals, AB, Uppsala, Sweden) containing Su- perose-12 or Superose-6 equilibrated with buffer B with 0.1 mM PMSF.

Assay for Kinesin-induced Gliding of Microtubule Axonemes-De- tergent-extracted Chlamydomonas axonemes were prepared as described by Witman (17) and stored for up to one week at 5 "C in the kinesin motility buffer of Porter et al. (18). It was important to isolate axonemes from stationary phase cultures to obtain axonemes that were quiescent and did not become reactivated in motility buffer in the absence of kinesin. No. 1% coverslips were cleaned in chromic acid, rinsed with distilled water, and air-dried. For motility assays, 10 pl of kinesin (10-500 pg/ml) was applied to the center of a coverslip, spread into a 1-cm diameter area with the tip of a pipette, allowed to adsorb for 30 s, and washed for 5 s with motility buffer. After removing excess buffer with a filter paper, axonemes (10 pl) and ATP (1 pl of a 10 mM stock) were added simultaneously, and the coverslip was mounted on a microscope slide and examined at room temperature by phase contrast microscopy on a Zeiss standard microscope or by video-enhanced DIC microscopy. For video-enhanced microscopy, we used a Leitz Orthoplan microscope fitted with a 100 X 1.32 NA NPL/Fluotar ICT objective and ICT condenser focused for Koehler illumination with a 100-watt halogen lamp. For video enhancement, the microscope was equipped with a Hamamatsu 2400 C camera and Newvicon tube and a Quantex DS 580 image processor. Images were recorded on a JVC CR-6060U 3/r-inch videocassette recorder in real time and analyzed directly on a TV screen. Magnifi- cation was calibrated by photographing the scale of a stage microm- eter.

To examine translocator activity of the 45-kDa fragment, 180 pg of kinesin in 0.6 ml of buffer B was digested with 4.5 pg of a- chymotrypsin for 15-45 min at room temperature, arrested with 5 mM PMSF and fractionated by fast protein liquid chromatography on Superose-6 as described above. The elution position of the 45-kDa fragment was determined by SDS-PAGE and silver staining. The peak tubes containing purified 45-kDa fragment a t 30 pg/ml were used for motility studies.

Electrophoresis-SDS-PAGE was performed by the method of Laemmli (19) on 12 or 15% polyacrylamide slab gels having an acrylamide to N,N'-methylenebisacrylamide weight ratio of 1OO:l. Gels were stained with 0.5% Coomassie Brilliant Blue R-250 or where indicated with silver nitrate (20).

One-dimensional peptide maps were prepared according to Cleve- land et al. (21) using 10% polyacrylamide gels containing a 300.8 weight ratio of acrylamide to N,N'-methylenebisacrylamide. The proteins used for peptide mapping were revealed by CuSO, staining as described by Lee et al. (22).

Biochemical Materials-a-Chymotrypsin (1-chloro-3-tosylamido- 7-amino-2-heptanone-treated, type VII), trypsin (type IX), subtilisin (bacterial protease, type VIII) were obtained from Sigma. Taxol was kindly provided by Dr. M. Suffness (Natural Products Branch, De- velopmental Therapeutics Programs, National Cancer Institute, Be- thesda, MD).

RESULTS

The Chymotryptic Cleavage Products of Kinesin-The effect of cy-chymotrypsin on kinesin is shown in Fig. 1. When incubated a t a kinesin to protease weight ratio of 200:l at 20 "C, the 62-kDa light chain of kinesin was completely degraded within 5 min. In contrast, the kinesin heavy chain was much more resistant to proteolysis and was substantially degraded only after prolonged digestion (not shown). During proteolysis the principal components of digestion included peptides with molecular masses of 74, 53, and 45 kDa. The 53-kDa fragment appeared early in digestion and then disap- peared (Fig. l ) , whereas the 74- and 45-kDa fragments became more obvious after more extensive digestion (Fig. 2). Thus, the size and amount of fragments occurring together in a given digest depend on the extent of digestion. The simulta- neous appearance of these peptides and the disappearance of the 120-kDa heavy chain suggested that the three peptides were derived from the 120-kDa kinesin heavy chain. This relationship is demonstrated below in more detail for the 45- kDa fragment.

t ime 0 5 10 20 40 80

205-

66-

45

29-

I- 7 4 * - LC

+ 53*

* 45 3c

1.0 1.0 12 -1.72.54.6 ATPase

FIG. 1. Kinetics of kinesin digestion with a-chymotrypsin. Digestion was performed at 20 "C in buffer B at an a-chymotrypsin to kinesin ration of 1:200 (w/w) and was stopped with 2 mM PMSF. Digests were analyzed by SDS-PAGE on 12% gels. Digestion time (in minutes) is indicated above the lanes. The locations and sizes (in kilodaltons) of selected molecular mass markers are shown on the left, and the locations of the heavy chain ( H C ) and light chain (LC) of kinesin and of the major kinesin chymotrypsin fragments of 74, 53, and 45 kDa are shown on the right. The relative ATPase activities of the digests are shown below the lanes. ATPase activity was measured in the presence of 1 mg/ml of microtubules. The activity of the control sample (undigested kinesin) was 1.2 pmol of Pi released per min/mg of kinesin.

Generation of a Kinesin Fragment with Enhanced Microtu- bule-binding Actiuities-In order to identify which chymotryptic peptides contained the microtubule-binding site of kinesin, we incubated partial digests with microtubules, sed- imented the polymers by centrifugation, and analyzed the peptides contained in the pellet by SDS-PAGE. For these experiments, kinesin was digested a t 37 "C a t a 50:l ratio of kinesin to chymotrypsin and arrested after 5 min with 2 mM PMSF. After this treatment the kinesin light chain and the 53-kDa fragment were difficult to observe, but the kinesin heavy chain and the 74- and 45-kDa fragments were conspic- uous (Fig. 2). Microtubules made of pure tubulin and stabilized with 20 p~ taxol were added to the kinesin digest, the mixture was supplemented with 2.5 mM PPPi to enhance the binding of kinesin fragments onto microtubules, and polymers were pelleted a t 150,000 X g for 30 min through a layer of 4 M glycerol containing buffer B supplemented with 2.5 mM PPPi, 0.1 mM PMSF, and 5 pM taxol. The polypeptide com- positions of the resulting pellet and supernatant are shown in Fig. 2. The pellet contained tubulin, the 45-kDa heavy chain fragment, some remaining intact kinesin heavy chain, and other trace components. From this it was also clear that the kinesin heavy chain could bind to microtubules in the absence of an intact light chain. Like intact kinesin, the 45-kDa fragment did not bind to microtubules in the presence of 2.5

A TPase-containing Fragment of Kinesin 591

1 2 3

120-

74-

T-

45-

FIG. 2. Binding of kinesin fragments to microtubules. Ki- nesin was digested with a-chymotrypsin (37 "C, 20 min, a-chymotrypsin to kinesin ratio of 1:50 (w/w)). The digest was mixed with an equal volume of microtubules made of pure tubulin and stabilized with 20 PM taxol. 2.5 mM PPPi was added and microtubules were pelleted in a SW 50.1 rotor a t 150,000 X g through a 4-ml cushion of 4 M glycerol in buffer B containing 5 p M taxol and 2.5 mM PPPi. The pellet and the supernatant were analyzed by SDS-PAGE. Lane 1, the digest; lane 2, pellet; lane 3, supernatant. The positions and corre- sponding molecular weights of the major polypeptides in the digest are indicated at the left. T indicates the position of tubulin.

mM ATP (data not shown), suggesting that the microtubule- binding site of the kinesin molecule was contained within the 45-kDa fragment. In contrast, most of the 74-kDa fragment remained in the supernatant and did not bind to microtubules. In other experiments not shown here, the 53-kDa fragment, which is generated by brief digestion with chymotrypsin, was observed to bind to and pellet with microtubules. However, the relationship between this early transient fragment and the more stable 45-kDa peptide is not known.

Interestingly, partial digestion of kinesin with other proteases such as trypsin or subtilisin resulted in fragments with electrophoretic mobilities similar to that of the 45-kDa fragment produced by chymotrypsin. The similar size of these protease-resistant fragments and the observation that the 45- kDa fragments produced by these enzymes also bound to microtubules in the presence of PPPi (data not shown) suggest that they represent similar domains of the molecule. In future studies it will be interesting to see if the protease-resistant fragment corresponds to one or more of the "heads" that have been detected by others by platinum shadowing.

The 45-kDa Fragment Contains ATPase Activity-The ATP-sensitive binding of the 45-kDa fragment to microtubules suggested that this fragment might contain the catalytic site of kinesin. To test this possibility, we studied the effect of a-chymotrypsin on ATPase activity. Fig. 1 shows that cleavage of the heavy chain stimulated ATPase activity and

that the degree of stimulation was roughly proportional to the amount of 45-kDa fragment contained in the digest. MgZ+- ATPase activities of the digest and of intact kinesin were fully dependent on the presence of microtubules. Stimulation of ATPase activity was also observed after proteolysis with trypsin or subtilisin (data not shown). Therefore, one of the peptides in the digest (most probably the 45-kDa fragment) was concluded to contain the catalytic site of the protein.

In order to identify and isolate this peptide, the digest was fractionated by Superose-12 chromatography on a HR 10/30 column. The elution profile, ATPase activity, and polypeptide composition of the fractions are shown in Fig. 3. Unlike the case for intact kinesin, the microtubule-dependent ATPase of the proteolytic digest was resolved into two peaks. The first peak contained a mixture of peptides with molecular masses of 40 to 100 kDa plus some undigested heavy chain. Fractions

-120

-45

B

32 A

FIG. 3. Purification of the 45-kDa fragment obtained by a- chymotrypsin digestion. Digestion was performed for 120 min a t 20 "C at a weight ratio of a-chymotrypsin to kinesin of 1:40. A, profile of the digest fractionated by Superose-12 chromatography on a HR 10/30 gel filtration column. The eluate was monitored continuously by absorption a t 280 nm (solid line) and the microtubule-activated Mg2"ATPase activities of individual fractions were determined (dashed lines). Arrows show the elution positions of bovine serum albumin ( a ) and a-chymotrypsin (c). B, SDS-PAGE of the fractions from the Superose-12 column. Positions of the kinesin heavy chain (120) and the 45-kDa fragment (45) are indicated on the right.

592 ATPase-containing Fragment of Kinesin

of the second peak contained the 45-kDa fragment and traces of the 53-kDa peptide. The ATPase activity across the fractions in the second peak was proportional to the amount of 45-kDa peptide. Since the fragments contained in the second peak eluted from the column after bovine serum albumin, the 45-kDa fragment was probably in the form of a monomer.

Gel filtration of the same kind of digest on a Superose-6 column gave similar results (data not shown). The first peak of ATPase activity contained a number of proteolytic fragments plus undigested heavy chain, and the second peak was enriched in the 45-kDa fragment. The second peak also contained several 20- to 35-kDa peptides, but no 53-kDa component (the only contaminant in the second peak after Superose- 12 chromatography) and no peptides larger than 45 kDa. Taken together the data of both chromatographic separations clearly show that the 45-kDa fragment is the ATPase-containing component.

To confirm this result and to show that the 45-kDa polypeptide can bind to microtubules in the absence of other parts of the kinesin molecule, we mixed microtubules (assembled from phosphocellulose-purified tubulin in the presence of 20 p~ taxol) with the 45-kDa fragment purified by Superose-12 chromatography and pelleted the complexes through a cushion of 4 M glycerol in buffer B containing 0.1 mM PMSF and 5 p~ taxol. Fig. 4 shows the polypeptide composition of the pellets and supernatants. The 45-kDa fragment pelleted with microtubules in the presence of PPPi, AMP-PNP, or in the absence of nucleotides but dissociated from microtubules in the presence of 2.5 mM ATP. ATPase assays showed that more than 85% of the activity pelleted with microtubules in the absence of nucleotides. These data clearly indicated that the 45-kDa fragment contains the ATPase site of the kinesin molecule.

Enzyme Kinetics-Since chymotryptic digestion stimulated overall ATPase activity, we compared the kinetic parameters of ATP hydrolysis of the 45-kDa fragment with those of undigested kinesin to determine if the ATPase activity of the fragment was greater than in the intact molecule. Double- reciprocal plots (Fig. 5) show that ATP hydrolysis by the fragment follows simple Michaelis kinetics, as was shown previously for the whole molecule (5). The fragment exhibited a K,,, for ATP of 70-100 p ~ , a Kapp for microtubules of 0.9- 1.2 p~ (expressed in terms of polymerized tubulin dimers), and a Vmax as high as 75-120 pmol of ATP hydrolyzed/mg of fragment/min. The fragment also resembled intact kinesin in exhibiting microtubule-independent ATPase activity when 2 mM Ca2+ was substituted for M$+ in the assay.

Table I compares the kinetic parameters of ATP hydrolysis by the 45-kDa fragment and intact kinesin. Data for the intact molecule were taken from our previous paper (1 1) and were independently confirmed in this study (data not shown). It is evident that the K,,, and Vmax of the fragment for ATP were approximately 10 and 20 times higher than the corre- sponding values for intact kinesin. As calculated from the value of VmaX (75-120 pmol/min/mg), the kat for the 45-kDa fragment (the maximum catalytic rate at saturating substrate) was 60-90 s-', four to five times higher than the value for intact kinesin.

The 45-kDa Fragment Is a Domain of the Kinesin Heavy Chain-Finally, to confirm that the 45-kDa fragment was really a part of the kinesin heavy chain as suggested by the digestion pattern in Fig. 1, we compared one-dimensional peptide maps of the 45-kDa fragment with those of the light (62-kDa) chain of kinesin (Fig. 6) using a-chymotrypsin and V8 protease from Staphylococcus aureus. It is evident that the 45-kDa fragment is distinct from kinesin light chain as judged

1 2 3 4 5 6 7 8 9

T-

45- - . - = - 0

FIG. 4. Cosedimentation of the purified 45-kDa fragment with microtubules. Lune 1, purified 45-kDa fragment; lane 2, supernatant and lane 6, pellet after centrifugation of the microtubule and fragment mixture in the absence of nucleotide; lane 3, supernatant and lane 7, pellet after centrifugation of the microtubule and fragment mixture in the presence of 1 mM AMP-PNP; lune 4, supernatant and lane 8, pellet after centrifugation of the microtubule and fragment mixture in the presence of 2.5 mM PPPi; lane 5, supernatant and lane 9, pellet after centrifugation of the microtubule and fragment mixture in the presence of 2.5 mM ATP. Centrifugation was performed in a SW 50.1 rotor a t 150,000 X g through a cushion of 4 M glycerol containing buffer B, 5 p~ taxol, and the appropiate nucleotides. Positions of the 45-kDa fragment and tubulin are indicated on the left. The gel was stained with silver nitrate.

by its greater sensitivity to the proteases and by the unique patterns of polypeptides produced. These results clearly dem- onstrate that the fragment is not a part of the light chain from which we conclude that it is derived from the kinesin heavy chain.

The 45-kDa Fragment Does Not Support Axoneme Glid- ing-To determine if the 45-kDa fragment contained translocator activity, we examined its ability to induce the gliding of Chlamydomonas axonemes on glass coverslips. We first examined the ability of intact kinesin to promote axoneme gliding. At concentrations of intact kinesin greater than 100 pg/ml almost all axonemes moved at a uniform velocity of 0.4 pm/s, but this value varied from experiment to experiment (range 0.2-0.8 pm/s in five separate experiments). The velocity of gliding that was observed (0.4 pm/s) was similar to that reported by Porter et al. (18), who used sea urchin flagellar axonemes and sea urchin egg kinesin prepared using AMP- PNP. Intact kinesin at 50 pg/ml also induced 80-90% of freshly prepared axonemes to glide, but no motility was observed with 12 pg/ml kinesin. Axonemes continued to glide in the preparations for 2 h or longer.

Unfractionated and fractionated preparations of digested kinesin were also tested for gliding activity. Kinesin at a concentration of 0.2 mg/ml was digested with chymotrypsin until -0.7 of the 120-kDa chain and all of the 62-kDa light chain were removed as determined by SDS-PAGE. Two preparations did not support motility; another preparation pro- moted gliding motions by some of the axonemes, but these

ATPase-containing Fragment of Kinesin 593

1 2 3 4 5 6 7 A 9 M 11 121314 A 1 T I

B

I I I I I I -0.8 -0.4 0.4 a8 "L vM-l

F 11 FIG. 5. Double-reciprocal plots of microtubule-activated

M$+-ATPase of the 45-kDa fragment as a function of ATP concentration ( A ) and microtubule concentration ( B ) . ATPase activity was measured in buffer B at 37 "C with saturating amounts of microtubules (1.0 mg/ml, A ) or ATP (2 mM, B) . The buffer system contains ATP regenerating components as described under "Mate- rials and Methods." Microtubule concentration is described in terms of the concentration of polymerized tubulin dimer (micromolar). The concentration of the 45-kDa fragment of kinesin was 0.1 pg/ml.

TABLE I Kinetic parameters of the ATP activities of kinesin and

its 45-kDa fragment The activity of kinesin fragment (0.1 pg/ml) was measured a t 37 "C

in buffer B (50 mM imidazole-HCI, pH 6.7, 3 mM MgCl2, 0.1 mM EDTA, 1 mM EGTA, 1 mM 6-mercaptoethanol). The values of Vmax, K,, and K., for kinesin fragment were taken from Fig. 5; the values for intact kinesin were taken from Kuznetsov and Gelfand (1986). Values of K,,, were determined for 4-40 pg/ml kinesin. Microtubule- activated Mg'"ATPase was determined in the presence of polymerized microtubules a t 1 mg/ml in buffer B supplemented with an ATP regenerating system. Kernindicates the tubulin concentration required for half-maximal ATPase activity and is expressed in terms of the concentration of polymerized tubulin.

Parameter Microtubule-

Me2'-ATPase activated Ca2+-ATPase

V,. (pmol/min/mg) Kinesin 4-6 1-2 ____

Fragment 75-120 0.7-1.5 K,,, for ATP ( p ~ ) Kinesin 10-12 -800

Fragment 75-100 - 1000 Kapp for microtubules Kinesin 12-14

(pmol of tubulin) Fragment 0.9-1.2

movements rapidly tapered off and stopped after 5 min, probably because of the low concentration of remaining 120- kDa chain (-60 pg/ml). The purified 45-kDa fragment of kinesin did not support motility at 30 pg/ml. We estimate that movements occurring at %O of the rate of gliding by intact kinesin would have been detected. Since the fragment mi-

20 40 80 100 200 400 FIG. 6. One-dimensional peptide maps of the 62-kDa kine-

sin light chain (lane I ) and the 45-kDa fragment (Zane 2) obtained by digestion with VS protease of S. aureus (lanes 3- 8 ) and a-chymotrypsin (lanes 9-14). Lanes with odd numbers are maps of the light chain; lanes with even numbers are maps of the 45-kDa fragment. The amounts of proteases in nanograms are shown below the lanes. The gel was stained with silver nitrate.

grated as a monomer on gel filtration columns, this concentration of fragment corresponds to a molar equivalent of intact kinesin of 120 pg/ml, which is within the range of concentrations giving optimal translocation activity. Since the fragment could have bound to the glass surface in an unfavorable orientation or lacked a domain essential for its attachment to glass, our observations are not yet conclusive, but we favor the idea that the 45-kDa fragment by itself is not capable of supporting motility.

DISCUSSION

A Fragment of the Kinesin Heavy Chain Contains Both ATPase and Microtubule-binding Activities-Proteolysis of kinesin by a-chymotrypsin causes the appearance of a 45- kDa fragment of the molecule, and simultaneously, a progres- sive activation of ATPase activity. We have also shown that the 45-kDa fragment binds to microtubules and demonstrated by Cleveland gel analysis that it is derived from the 120-kDa heavy chain. Therefore, both the ATPase and microtubule- binding sites of the molecule are located on the same 45-kDa domain of the heavy chain. These results agree with the data of Gilbert and Sloboda (23), Penningroth et al. (24) and Bloom et al. (12), who showed that a nucleotide-binding site, and therefore possibly the site for ATPase activity, is located on the kinesin heavy chain.

The fact that proteolysis stimulates ATPase and that the amount of ATPase activity varies in different kinesin preparations suggests that the variation in ATPase content could be due to proteolysis. Of particular interest is the fact that kinesin isolated with PPPi contains high levels of microtubule-activated Mg2"ATPase activity (ll), whereas preparations made with AMP-PNP do not (1, 12, 24-26). Typical values for ATP hydrolysis have been reported at 3-5 (11, 27) and 0.007-0.15 pmol/min/mg (1,12), respectively. Proteolytic activation of ATPase does not explain the difference in ATPase activity, however, because the kinetic parameters of ATP hydrolysis exhibited by intact kinesin and kinesin fragment are distinct and easily distinguished from one another. One idea discussed previously that still needs to be examined is that the differences may in some way be due to differences in the inhibitory effects of PPPi and AMP-PNP (12). This

594 ATPase-containing Fragment of Kinesin

idea seems to be supported by the recent result of Kachar et al. (27), who found that the kinesin-like protein from Acan- thameba in the absence of AMP-PNP contained high ATPase activity. However, Cohn et al. (26) showed directly that exclu- sion of AMP-PNP from the buffers used to isolate sea urchin kinesin did not result in increased ATPase activity. Therefore, the cause of the variability in ATPase activity still remains unclear.

Comparison of the Kinetic Properties of Kinesin with Those of Dynein and Myosin-Previous biochemical studies suggested that the mechanism of action of kinesin as a force- transducing protein is similar yet distinct from those of dynein and myosin. In the absence of ATP, kinesin forms complexes between beads or vesicles and microtubules (5,6,28) reminis- cent of the rigor complexes formed between dynein and microtubules or myosin and actin filaments. In addition, the Mg2”ATPase of kinesin is stimulated 60- to 120-fold by microtubules (11,27) in a manner analogous to the activation of dynein ATPase by microtubules or myosin ATPase by actin filaments. There are, however, differences in the re- sponses of the translocators to AMP-PNP, a nonhydrolyzable analogue of ATP. AMP-PNP induces the binding of mem- brane vesicles to microtubules in rigor-like complexes that can only be dispersed by high concentrations of ATP. In contrast, AMP-PNP dissociates actomyosin at dilute (but not at high) concentrations of actomyosin (29) and appears to dissociate dynein from outer doublet microtubules of axonemes (30) but cannot relax the rigor state in ATP-depleted axonemes (31). The situation is actually complex, and the reader is referred to an excellent discussion by Johnson (32) for further details. Despite these differences in response to nucleotide analogues, it appears that the mechanisms of the three enzymes are related. The comparative analysis of the kinetics of ATP hydrolysis by intact kinesin and kinesin fragment described in the present paper increases our under- standing of this relationship still further.

In a recent review comparing the mechanisms of action of dynein and myosin, Johnson (32) noted that the 100- to 200- fold difference in the rate of product release from dynein and myosin is reflected in a 100-fold difference in their K,,, values for ATP, which are 1.7 and 0.012 p ~ , respectively. Values of K, are not influenced by the rates of substrate binding, which are rapid and similar in magnitude for both enzymes. We may therefore compare the K, values of intact kinesin and kinesin fragment (shown in Table I) to estimate whether proteolysis increased ATPase activity by increasing the rate of product release from the fragment. Viewed in this way, the observed &fold increase between intact kinesin (10-12 p ~ ) and kinesin fragment (75-100 p ~ ) might account for the increased rate of product release by the fragment. Interestingly, the Kapp for microtubule binding decreases from 12 to 14 pM for intact kinesin to 0.9-1.2 p~ for kinesin fragment, suggesting that the increase in ATPase activity is related to an accompanying increase in the affinity of the fragment for microtubules. Our estimation of a very low Kapp for kinesin binding to microtubules agrees with the value previously reported by Kachar et al. (27). More work is required to examine the validity of these assumptions.

Uncoupling of ATPase and Translocation Activities May Stimulate ATPase Activity-Comparative studies of the kinetics of ATP hydrolysis by intact kinesin and kinesin fragment show that ATP hydrolysis is closely linked to microtubule binding and suggest that there is an overall similarity in the ATPase mechanisms of kinesin and the other motility ATPases, dynein, and myosin. However, an important difference between kinesin and other ATPases is that proteolytic

digestion activates kinesin ATPase but does not affect the ATPase activities of dynein or myosin (33-35). At present an explanation for this difference in behavior is not available. We propose that cleavage and removal of the fragment from other parts of the heavy chain may uncouple the ATPase and translocation activities of the molecule, resulting in futile cycles of ATP hydrolysis and activation of the ATPase. If true, the results would suggest that mechanochemical activity of kinesin, unlike that of myosin (35), requires other parts of the molecule in addition to the ATPase and microtubule- binding sites contained in the domain occupied by the fragment.

The generation and analysis of specific proteolytic fragments of dynein and myosin have proved useful in examining a variety of structure-function relationships for dynein and myosin. Dynein fragment A produced by digestion with trypsin (33) and fragments produced by vanadate-sensitive pho- tocleavage (36) have been used to map the site of ATPase activity on the dynein heavy chain. The isolation of heavy meromyosin and subfragment 1 from myosin is particularly noteworthy, because these proteolytic fragments made it pos- sible to study the myosin ATPase mechanism by conventional methods of enzymology (37) and examine physical and struc- tural aspects of myosin-actin filament interactions (38). We are optimistic that the 45-kDa fragment of kinesin will also prove useful for examining the mechanism, structure, and function of kinesin ATPase. If proteolytic cleavage of kinesin really does uncouple ATPase and translocation activities, as we suspect, this fragment may be of particular interest for future studies of mechanochemical coupling in vesicle transport.

Acknowledgments-We thank Professor A. S. Spirin for his support of this work. We also wish to thank Dr. A. S. Girshovich for making available his FPLC equipment, Dr. G. Witman for materials and advice in preparing Chlamydomonas axonemes, and Drs. S. GIlbert, J. Scholey, M. Sheetz, and R. Sloboda for advice in isolating kinesin and designing an assay for kinesin motility, and Dr. M. Suffness for the gift of taxol. We give special thanks to G. A. Kuznetsova and K. T. Wallis for their excellent technical assistance.

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Isolation of a 45-kDa Fragment from the Kinesin Heavy Chain with