Essential kinesins: characterization of Caenorhabditis elegans KLP-15.

Abstract

Kinesins form a superfamily of molecular motors involved in cell division and intracellular transport. Twenty kinesins have been found in the Caenorhabditis elegans genome, and four of these belong to the kinesin-14 subfamily, i.e., kinesins with C-terminal motor domains. Three of these kinesin-14s, KLP-15, KLP-16, and KLP-17, form a distinct subgroup in which KLP-15 and KLP-16 are more than 90% identical and appear to be related by a relatively recent gene duplication. They are essential for meiotic spindle organization and chromosome segregation, and are mostly expressed in the germline. With 587 amino acids each, they are among the smallest kinesins known. Using bacterially expressed KLP-15 constructs with different length extensions preceding the motor domain, we have determined in vitro the following characteristic properties: ATPase activity, microtubule binding, oligomeric state, microtubule gliding activity, and direction of movement. The constructs exhibit a monomer-dimer equilibrium that depends on the length of the predicted alpha-helical coiled-coil region preceding the motor domain. The longest construct with the complete coiled-coil domain is a stable dimer, and the shortest construct with only seven amino acids preceding the motor domain is a monomer. In microtubule gliding assays, the monomer is immobile whereas the fully dimeric KLP-15 construct supports gliding at 2.3 microm/min and moves toward microtubule minus ends, like other members of the kinesin-14 subfamily studied to date.

Full text

Page 1

Essential Kinesins: Characterization of Caenorhabditis elegans KLP-15†
Gautier Robin,‡,§Salvatore DeBonis,‡Aure ´lie Dornier,|Giovanni Cappello,|Christine Ebel,‡Richard H. Wade,*,‡
Danielle Thierry-Mieg,⊥and Frank Kozielski*,‡
Institut de Biologie Structurale, UMR 5075 CEA/CNRS/UJF, 41 rue Jules Horowitz, 38027 Grenoble Cedex 01, France,
Laboratoire de Physico-Chimie, Institut Curie, UMR 168 CNRS/Institut Curie, 11 rue Pierre et Marie Curie,
75231 Paris Cedex 05, France, and National Center for Biotechnology Information, National Library of Medicine,
National Institutes of Health, Building 38A, 8600 RockVille Pike, Bethesda, Maryland 20894
ReceiVed August 26, 2004; ReVised Manuscript ReceiVed February 15, 2005
ABSTRACT: Kinesins form a superfamily of molecular motors involved in cell division and intracellular
transport. Twenty kinesins have been found in the Caenorhabditis elegans genome, and four of these
belong to the kinesin-14 subfamily, i.e., kinesins with C-terminal motor domains. Three of these kinesin-
14s, KLP-15, KLP-16, and KLP-17, form a distinct subgroup in which KLP-15 and KLP-16 are more
than 90% identical and appear to be related by a relatively recent gene duplication. They are essential for
meiotic spindle organization and chromosome segregation, and are mostly expressed in the germline.
With 587 amino acids each, they are among the smallest kinesins known. Using bacterially expressed
KLP-15 constructs with different length extensions preceding the motor domain, we have determined in
vitro the following characteristic properties: ATPase activity, microtubule binding, oligomeric state,
microtubule gliding activity, and direction of movement. The constructs exhibit a monomer-dimer
equilibrium that depends on the length of the predicted R-helical coiled-coil region preceding the motor
domain. The longest construct with the complete coiled-coil domain is a stable dimer, and the shortest
construct with only seven amino acids preceding the motor domain is a monomer. In microtubule gliding
assays, the monomer is immobile whereas the fully dimeric KLP-15 construct supports gliding at 2.3
µm/min and moves toward microtubule minus ends, like other members of the kinesin-14 subfamily studied
to date.
Kinesins form a large superfamily of eukaryotic molecular
motors which interact with microtubules and participate in
intracellular transport and cell division. They transform
chemical energy from ATP hydrolysis into mechanical work,
allowing them to move rapidly and in a specific direction
along microtubules (1). Twenty genes encoding kinesin-like
proteins have been identified in the Caenorhabditis elegans
genome (2, 3), and many of these have mammalian ortho-
logues. The in vivo function of several nematode kinesins
has been extensively investigated through observations of
loss of function phenotypes induced by mutation or RNA
interference (4-12). Although most kinesins have their
∼320-amino acid motor domain close to their N-terminus,
there is an important subfamily with the motor domain near
the C-terminus of the polypeptide chain; in the proposed new
nomenclature, this subfamily is now called the kinesin-14
family (13, 14). There are four kinesin-14s in C. elegans,
KLP-3,1KLP-15, KLP-16, and KLP-17, with the last three
forming a distinct subgroup in proximity to Drosophila
melanogaster ncd and Saccharomyces cereVisiae Kar3 (14).
KLP-15 and KLP-16 are practically identical proteins with
587 amino acid residues each [ClustalW alignments (15) give
91.1% identical, 4.3% strongly similar, and 2.0% weakly
similar amino acids for the full-length proteins]. The
complete mRNAs for klp-15 and klp-16 are also strikingly
similar. They are 93% identical along their entire length
(including the UTRs), implying that they may be related by
a relatively recent gene duplication. They are on the same
chromosome (chromosome 1), as is usual for gene duplica-
tions, and are less than four megabases apart. The positions
†This research has been funded by grants from ARC (Association
pour la Recherche sur le Cancer, Contracts 4210 and 5917) and the
CNRS (Centre National de la Recherche Scientifique). G.R. acknowl-
edges support from the Ministe `re de l’Education Nationale, de la
Recherche et de la Technologie (MENRT), and the Ligue Nationale
contre le Cancer.
* Towhomcorrespondence
Frank.Kozielski@ibs.fr (F.K.) or wade@ibs.fr (R.H.W.). Telephone:
0033-4-3878-4024. Fax: 0033-4-3878-5494.
‡UMR 5075 CEA/CNRS/UJF.
§Current address: Institute for Molecular Bioscience, University of
Queensland, Brisbane, Queensland 4072, Australia.
|UMR 168 CNRS/Institut Curie.
⊥National Institutes of Health.
shouldbeaddressed.E-mail:
1Abbreviations: AMPPCP, adenosine 5′-(?-methylene)triphosphate;
?-ME, ?-mercaptoethanol; CCD, charge-coupled device; DVCAM,
digital video camera; DTT, dithiothreitol; EGTA, ethylene bis-
(oxyethylenenitrilo)tetraacetic acid; IPTG, isopropyl ?-D-thiogalacto-
pyranoside; KLP, kinesin-like protein, the equivalent gene is given in
small letters; MT(s), microtubule(s); NA, numerical aperture; Ncd, non-
claret disjunctional; NIH, National Institutes of Health; Ni-NTA,
nickel-nitriloacetic acid; PCR, polymerase chain reaction; PEM,
PIPES, EGTA, magnesium buffer; PIPES, piperazine-N,N′-bis(2-
ethanesulfonic acid); PMSF, phenylmethylsulfonyl fluoride; RNAi,
RNA interference; SDS-PAGE, sodium dodecyl sulfate-polyacryl-
amide gel electrophoresis; UTR, untranslated region; VE-DIC, video-
enhanced differential interference contrast.
6526
Biochemistry 2005, 44, 6526-6536
10.1021/bi048157h CCC: $30.25 © 2005 American Chemical Society
Published on Web 04/06/2005

Page 2

and lengths of the introns and exons in the klp-15 and klp-
16 genes are identical, except for a single base pair insertion
and a single base pair deletion in two introns. The homology
even extends to the promoter area, with 89% identity over
the 131 bp immediately upstream of the genes. In practice,
the sequence similarity of the two genes will prevent selective
targeting of one or the other by RNA interference, cosup-
pression, or RNA hybridization.
The most obvious consequences of RNAi with either klp-
15 or klp-16 are defects in meiosis (16-19) [see the movies
at http://www.RNAi.org (20)]. Normally, C. elegans oocytes
are arrested in the prophase of meiosis I, and this block is
released at fertilization when the diploid and haploid products
of meiosis I and II are ejected outside of the embryo as polar
bodies. Inhibition of KLP-15/KLP-16 function, obtained by
RNAior cosuppression, results in failure in meiotic spindle
assembly, and consequently leads to defects in meiotic
chromosome segregation. The meiotic prophase nuclei appear
to be normal, and homologues are paired and attached by
chiasmata; however, the polar bodies are abnormal in number
or size, and correlatively the female pronucleus may also be
of abnormal size. Probably as a result of aneuploidy, multiple
nuclei sometimes form in one, two, and four cell embryos.
Finally, an excess of males (X0) is observed among the few
survivors, and this also indicates defective meiotic chromo-
some segregation. Interestingly, these defects are closely
similar to those of a number of other essential genes that
form a functional cluster that is active during meiosis and
mitosis, in particular, cyclin B and the Drosophila abnormal
spindle homologue. In situ hybridization shows that klp-15
and/or klp-16 mRNAs are concentrated in the germline at
all stages, from L1 to adult (Figure 1). In L3, L4, and very
young adults, transcripts accumulate in a specific region of
the gonad, dorsal, and preturn, corresponding to the prophase
of meiosis I, after the synaptonemal complex has been
assembled, during pachytene. In older adults, the transcripts
spread to the ventral gonad and are also present in large
amounts in very early embryos. We conclude that the klp-
15 and klp-16 genes are essential for acentriolar meiotic
spindle formation and proper chromosome segregation.
KLP-15 and KLP-16 are predicted to have three structural
domains: an N-terminal globular tail domain (residues
Met1-Pro148), an R-helical coiled-coil region (residues
Ser149-Leu246), potentially leading to dimerization or
higher oligomers, and a globular C-terminal motor domain
(Ile249-Val572), including the ATP-binding and MT-
interaction sites. Previous studies on kinesin-1s, the conven-
tional kinesins with the motor domain close to the N-termi-
nus, have indicated that the R-helical coiled-coil domain is
important for dimerization and strongly influences the kinetic
properties of these kinesins. Curiously, the dimerization of
the kinesin-14 subfamily has never been studied in any detail,
and because of their small size, KLP-15/16 kinesins might
possibly function as either dimers or monomers. Conse-
quently, to initiate studies of these kinesins, we have cloned,
expressed, and purified several KLP-15 constructs covering
the motor domain and various lengths of the putative coiled-
coil domain. These constructs have a His tag at their
N-termini to facilitate purification and motility studies. We
have examined the oligomeric state of these proteins, their
in vitro ATPase kinetics, their microtubule binding, and
finally their motility and directionality as determined by
microtubule gliding observations.
EXPERIMENTAL PROCEDURES
Materials. The Rapid Excision Kit, the QuikChange Site-
Directed Mutagenesis Kit, and Epicurean Coli XL10 Gold
cells were obtained from Stratagene. Synthetic oligonucle-
otides were bought from MWG-Biotech AG. The Taq PCR
Master Mix Kit for PCR, the plasmid DNA purification kit,
the Ni-NTA conjugate, and Ni-NTA were purchased from
Qiagen. Restriction enzymes were bought from NEB Biolabs.
The Rapid DNA Ligation Kit was from Boehringer. The
enzymes for kinetic assays and anti-His tag antibodies for
microtubule gliding assays (catalog no. H1029) were from
Sigma. Cover slips (superfrost plus, 22 mm × 75 mm ×
1.0 mm, catalog no. 041300) for gliding assays were bought
from Menzel Glazer. Beef tubulin for gliding assays and
rhodamine-labeled tubulin were purchased from Cytoskel-
eton.
Cloning of KLP-15 Constructs. The cDNA clone yk
1466a11, encoding the entire KLP-15 protein, was obtained
from Y. Kohara (National Institute of Genetics, Mishima,
Japan). The insert was excised from the viral genome (λ
ZAPII phage suspension) and cloned into the pbluescript
vector using XPORT and XLOLR phages as described by
the manufacturers. Potential monomeric and dimeric KLP-
15 proteins were designed by aligning the protein sequence
with D. melanogaster ncd. Unique restriction sites were
introduced into the forward (NheI) and reverse primers (NotI)
to allow subsequent cloning into Escherichia coli pET
expression vectors. The corresponding DNA constructs were
synthesized using the polymerase chain reaction. The fol-
lowing forward primers were used: A (klp-15239-587), 5′-
GAGTTGAGAAAGTTGGCTAGCGATGTTGTCGATT-
TA-3′; B (klp-15215-587), 5′-AAAGTGGAGGAGTGCGCTA-
GCTATCGTGTGCACAAC-3′; C (klp-15200-587), 5′-TCACT-
GCAAGATCAAGCTAGCACGCTGAAAGAAGTC-3′; D
(klp-15185-587), 5′-CAAATCCTGGATGGCGCTAGCGAAG-
GAGCGGATCGC-3′; and E (klp-15148-587), 5′-CAGAAGC-
CTATTCTCGCTAGCAAGATGGCGCTTCTT-3′.
FIGURE 1: Expression patterns of klp-15 and/or klp-16 genes during
development. Images obtained by in situ hybridization to klp-15/
klp-16 mRNA. The mRNA for these two kinesin-14s is indistin-
guishable and is enriched in the two germline cells in L1 larvae
(arrows). From the early L4 larval stage to young adults, expression
is maximal in the late pachytene of meiosis I. It appears to spread
to the diakinesis region in older adults. The V indicates the vulva
and labels the ventral side of the animal.
Characterization of C. elegans KLP-15
Biochemistry, Vol. 44, No. 17, 2005 6527

Page 3

The following reverse primer was used in all cloning
strategies: O, 3′-TACAGATACTTGATCCGCCGGCGAA-
GATAAGATTGTAAA-5′. The resulting PCR products were
digested using NheI and NotI restriction enzymes, gel
purified, isolated, and ligated into expression vectors, previ-
ously restricted with the same enzymes. Fragment B was
cloned into pET17b (ampicillin resistant), whereas fragments
A and C-E were ligated into pET28a, which carries the
resistance for kanamycin. Fragments A and C-E automati-
cally carried a coding sequence for six histidine residues at
the N-terminus of the protein, since pET28a carries a start
codon and a His tag coding sequence upstream of the NheI
restriction site. Fragment B required a second cloning step
for the incorporation of the histidine tag into the coding
sequence. For this purpose, the three plasmids were digested
with NheI and the resulting sticky ends dephosphorylated.
The following forward and reverse primers encoding six
histidines were ligated into the linearized vector:
CTAGCCACCACCACCACCACCACCCATGGG-3′ and 3′-
GGTGGTGGTGGTGGTGGTGGGTACCCGATC-5′. Posi-
tive clones containing the desired insert were identified by
double restriction with NheI and NotI, and the correctness
of the cloning strategy was confirmed by DNA sequencing
of all the constructs.
Expression and Purification of Protein Constructs. All
KLP-15 proteins were purified at 4 °C using the same batch
protocol. We optimized the expression of the different
constructs by testing several E. coli strains, IPTG concentra-
tions, and expression temperatures and times. Cells were
harvested, frozen in liquid nitrogen, and stored at -80 °C.
For protein purification, the cells were thawed and resus-
pended in buffer A [50 mM NaH2PO4/Na2HPO4(pH 7.5), 4
mM MgCl2, 300 mM NaCl, 10% glycerol, 5 mM imidazole
(pH 7.5), 0.1 mM Na-EGTA, and 5 mM ?-ME]. Cells were
lysed twice in the presence of 1 mM PMSF, 1 mM ATP,
and 40 µg/mL DNAse using a French press. The lysate was
centrifuged at 48000g for 20 min, and the supernatant was
incubated for 1 h with 5 mL of Ni-NTA resin previously
equilibrated with buffer A. The Ni-NTA batches were
washed at least four times with buffer B [20 mM PIPES
(pH 7.0), 4 mM MgCl2, 200 mM NaCl, 5% glycerol, 40
mM imidazole (pH 7.5), and 5 mM ?-ME]. The Ni-NTA
resin with bound KLP-15 protein was poured into small
columns and the protein eluted with buffer C [20 mM PIPES
(pH 7.0), 4 mM MgCl2, 30 mM NaCl, 5% glycerol, 200
mM imidazole (pH 7.5), and 5 mM ?-ME], taking fractions
of 1 mL. The fractions containing the protein were concen-
trated to ∼20 mg/mL using a CENTRICON-30 and applied
to a gel filtration column, previously equilibrated in buffer
D [30 mM PIPES (pH 7.0), 1 mM MgCl2, 30 mM NaCl, 1
mM Na-EGTA, and 1 mM DTT]. The peak fraction was
pooled, concentrated, frozen in liquid nitrogen, and stored
at -80 °C until it was used.
The protein concentration was measured by using the
theoretical extinction coefficients calculated from the primary
protein sequence using ProtParam (21) (http://au.expasy.org/
tools/protparam.html) (20 340 M-1cm-1for KLP-15239-587,
27 310 M-1cm-1for KLP-15215-587, 21 620 M-1cm-1for
KLP-15200-587, 21 620 M-1cm-1for KLP-15185-587, and
21 620 M-1cm-1for KLP-15148-587). To release bound ADP
that also contributes to the extinction coefficient (22), 100
5′-
µL of each KLP-15 protein was precipitated using 7%
perchloric acid and incubated for 5 min on ice. We assume
that no cystine (S-S) is present in KLP-15, and therefore,
cysteine residues present in the primary sequence do not
contribute to the extinction coefficient. After centrifugation,
the pellets were resuspended in 6.0 M guanidine hydrochlo-
ride and 200 mM phosphate buffer (pH 6.5) and the
absorption was measured at 280 nm. Further analysis of the
supernatants showed that all the protein was precipitated,
and the ADP that was released corresponded to one ADP
per site (data not shown).
The Bradford method (23) was used to calibrate each
construct with known concentrations measured with the
method described above. Five replicates per construct were
averaged. The tubulin concentration was determined by the
absorbance at 275 nm in 6 M guanidine hydrochloride, using
an extinction coefficient of 1.03 mL mg-1cm-1(24). The
concentration is expressed in tubulin dimers (110 kDa).
The coupled ATPase activity test with ATP regeneration
(24) was used to study the enzymatic characteristics of KLP-
15. The basal ATPase activity was measured at KLP-15
concentrations between 1 and 3 µM, and the MT-activated
ATPase activity was determined at a protein concentration
of ∼0.5 µM.
Analytical Ultracentrifugation. A Beckman Optima XL-I
analytical ultracentrifuge equipped with an An-60 Ti rotor
was used to perform sedimentation velocity experiments at
60 000 rpm (∼300000g) at a temperature of 10 °C in 20
mM PIPES (pH 7.3), 30 mM NaCl, 1 mM MgCl2, 1 mM
Na-EGTA, 1 mM DTT, 1 mM NaN3, and 10 µM MgATP.
The five different KLP-15 proteins were investigated at
various concentrations. The cells were equipped with quartz
windows with, for protein concentrations between 0.5 and 1
mg/mL, 12 mm optical path central pieces of 440 µL and,
for higher protein concentrations, 3 mm optical path central
pieces of 110 µL. Before each run, the diluted protein was
incubated overnight at 4 °C in the cell, to reach the
dissociation equilibrium. For each construct and measure-
ment, the wavelength was individually adjusted in the range
of 265-272 nm. The Sedfit program package (http://
www.analyticalultracentrifugation.com) was used to analyze
the sedimentation profiles in terms of either a continuous
distribution of the sedimentation coefficient c(s) (25) or one
or two discrete, noninteracting, species (26). Sedfit also
incorporates a systematic noise evaluation procedure (27).
We used the Sednterp software to estimate the partial specific
volumes of the KLP-15 constructs (0.7240-0.7258 mL/g)
and the density (1.00183 mL g-1) and viscosity (0.013207
mPa s) of the solvent (Sednterp version 1.01; developed by
D. B. Haynes, T. Laue, and J. Philo; http://www.bbri.org/
RASMB/rasmb.html), which were used to calculate normal-
ized coefficients (s20,w) from the experimental sedimentation
coefficients (s). For the c(s) analysis, we typically used 70
experimental profiles obtained every 5 min. We considered
200 particles with a frictional ratio (f/f0) of 1.5, with
sedimentation coefficients between 1 and 6 S, and used a
grid of 500 radial points and a confidence ratio of 0.68 for
the regularization procedure. The c(s) distributions were then
fitted to one or two Gaussians to provide values for the
sedimentation coefficients and for the relative concentrations
of the species. In our specific experiments, we have to
consider essentially two species, namely, monomers and
6528 Biochemistry, Vol. 44, No. 17, 2005
Robin et al.

Page 4

dimers. These are considered to be noninteracting if there is
no monomer association or dimer dissociation over the time
scale of the experiment. Analysis in terms of the one or two
noninteracting component model gives direct fits for the
values of s, Mw, and concentration. Equilibrium dissociation
constants, Kd, were calculated using eq 1 to fit the measured
dimer percentage (% dimer) as a function of the total protein
concentration, Ctotal.
This equation is obtained from the following definitions
where subscripts indicate total, monomer, and dimer con-
centrations:
Pelleting Assays. Pelleting assays were performed as
described previously (28). Tubulin and KLP-15 concentra-
tions were determined as described above. Experiments in
the presence of either ADP or AMPPCP were carried out at
a KLP-15 concentration of 4 µM and with tubulin concentra-
tions between 0 and 40 µM. The final volume of the reaction
solution was 48 µL in PEM buffer supplemented with 1 µM
taxol, 4 mM magnesium acetate, and 3 mM nucleotide (ADP
or AMPPCP). KLP-15215-587precipitated at higher AMPPCP
concentrations. To improve stability, KLP-15215-587 was
stored in the presence of at least 50 µM MgATP. For the
pelleting experiment, the protein was diluted five times to
give a final MgATP concentration of 10 µM. Thus, experi-
ments performed in the “absence of nucleotide” contain a
maximal MgATP concentration of 10 µM. We used NIH
image (http://rsb.info.nih.gov/nih-image) for the quantitative
analysis of bound and unbound kinesin concentrations.
Protein concentrations were determined from the intensity
of the bands in the SDS-PAGE gel by calibrating against a
range of known KLP-15215-587concentrations.
The binding stoichiometry of the monomeric KLP-
15215-587 and microtubules, along with the dissociation
constant, was determined by plotting the KLP-15215-587
concentration cosedimenting with microtubules as a function
of total microtubules (expressed as tubulin dimer concentra-
tion). Mutual depletion was taken into account by fitting the
plot with the following equation (28, 29):
where N/N0 is a normalizing factor (corresponding to the
maximum fraction of kinesin binding to microtubules), A )
Kd+ Mt0+ N0, MtN is the concentration of KLP-15 bound
to microtubules, N is the maximum concentration of bound
KLP-15, N0 is the total KLP-15 concentration, Mt0 is the
total tubulin concentration, and Kdis the apparent dissociation
constant.
In Vitro Microtubule Gliding Assays. Gliding assays were
performed by binding purified KLP-15 via the N-terminal
His tag to anti-His antibodies attached to the glass coverslip
of the perfusion chambers. KLP-15 was transferred into
buffer E [10 mM imidazole (pH 7.5), 2 mM magnesium
formate, 50 mM NaCl, 1 mM Na-EGTA, 1 mM DTT, and
4 mM MgATP] by gel filtration. Then 20 µL of anti-His tag
antibodies at a concentration of ∼10 µM in buffer E was
injected into the perfusion chamber and incubated for 5 min.
The chamber was washed with 20 µL of buffer E, and then
20 µL of KLP-15 at 30 µM in buffer E was injected and
incubated for 10 min. After the chamber had been washed
with 20 µL of buffer E, MTs were injected at a concentration
of 1 µM in buffer E. Finally, the chamber was washed with
buffer E supplemented with 10 µM paclitaxel and placed
on the microscope to record MT gliding.
We used a VE-DIC microscope equipped with a 1.4 NA
objective with a magnification of 100× and a Cohu CCD
camera. The microscope was maintained at 20 °C in a
temperature-controlled room. Image treatment (background
subtraction, signal-to-noise improvement) was performed
using an Argus-20 Hamamatsu image processor. MT move-
ment was recorded on digital DVCAM recording media. A
Neurospora crassa conventional kinesin construct, Nkin460-
GST (30), whose gliding velocity has been previously
established (31), was used as a positive control. This protein
is stable at room temperature. Nkin460GST was purified as
described previously (31).
MT gliding velocities V (micrometers per second) were
determined from the video recordings using NIH image
software with some in-house modifications of existing
macros. The coordinates of selected MT extremities were
obtained using the computer cursor and together with the
acquisition times of successive images were exported into
Excel. Gliding velocities V were calculated using the
following equation:
where shift ) ?[(x2- x1)2+ (y2- y1)2] is the displacement
calculated from the pixel coordinates (x1,y1) and (x2,y2) of a
MT extremity in image numbers N1 and N2, respectively.
The recording rate was 25 images per second, giving a time
delay of (N2- N1)/25 between images N1and N2. The scale
factor E converts the shift from pixels into micrometers (10
µm corresponds to 97 pixels both horizontally and vertically
on the CCD camera).
The direction of movement along microtubules was
determined by a modified protocol, originally described by
Hyman, using microtubules with rhodamine-labeled minus
ends (32, 33).
RESULTS
Five KLP-15 proteins were expressed and purified (Figure
2a,b). As shown in Table 1, the calculated molecular masses
derived from the KLP-15 primary sequence agree with the
masses measured by MALDI mass spectrometry. Using gel
filtration, we estimated that KLP-15239-587 is a monomer
whereas KLP-15215-587, a construct including a longer portion
of the predicted coiled-coil domain (Figure 2c), elutes with
a double peak, possibly indicating a monomer-dimer
equilibrium (data not shown). The longer KLP-15 constructs
appear to be dimers. The oligomeric state of the five KLP-
15 constructs was examined in more detail by sedimentation
velocity experiments as described below.
Hydrodynamic Properties. A typical set of measured
sedimentation profiles and calculated fits is shown in Figure
% dimer )
100[Kd+ 4Ctotal- (Kd
2+ 8KdCtotal)1/2]/(4Ctotal) (1)
Kd) Cmonomer
2/Cdimer; Ctotal) Cmonomer+ 2Cdimer;
% dimer ) 100(2Cdimer/Ctotal)
MtN ) (N/2N0)[A - (A2- 4N0Mt0)1/2] (2)
V ) 25E × shift/(N2- N1) (3)
Characterization of C. elegans KLP-15
Biochemistry, Vol. 44, No. 17, 2005 6529

Page 5

3. Figure 4a-e shows the distribution of sedimentation
coefficients of the KLP-15 constructs for different protein
concentrations. The shortest and longest constructs (KLP-
15239-587and KLP-15148-587, respectively) show single peaks,
with s20,wvalues of 3.0 and 4.3 S, respectively, the positions
of which do not change significantly in the investigated range
of protein concentrations. This suggests that the shortest
construct with 348 amino acid residues (KLP-15239-587) is
monomeric and the longest with 439 residues (KLP-15148-587)
is dimeric. The sedimentation coefficient (s20,w) 3.0 S) for
KLP-15239-587is consistent with values previously reported
for kinesin monomer constructs such as D. melanogaster ncd
(MC6) with 367 residues (3.31-3.7 S) (28, 34) and
conventional kinesins with 340 (3.25 S) and 341 residues
(2.9 S) (35-36). The sedimentation coefficient (s20,w) 4.3
S) of KLP-15148-587can be compared to the values of 4.9
and 5.0 S found for dimeric ncd MC1 with 491 residues
(there are 13 additional residues at the N-terminus due to
the expression system) and with the values of 5.18 and 5.06
S for conventional kinesin constructs with 392 and 401
FIGURE 2: C. elegans KLP-15 constructs. (a) The N-terminal tail domain of KLP-15 (residues Met1-Pro148) is followed by a continuous
R-helical region (residues Ser149-Leu246) predicted to form a coiled coil leading to dimerization. The C-terminal motor domain (residues
Ile249-Val572) contains the ATP-binding and MT-interacting sites. The KLP-15 proteins investigated contain the motor domain and
varying numbers of residues in the R-helical region, as shown. (b) SDS-PAGE of purified KLP-15 proteins: lane 1, standard protein
markers; lane 2, KLP-15239-587; lane 3, KLP-15215-587; lane 4, KLP-15200-587; lane 5, KLP-15185-587; and lane 6, KLP-15148-587. (c) Coiled-
coil regions in KLP-15 as predicted by COIL (50) and PAIRCOIL (51).
Table 1: Summary of the Physical Properties of KLP-15 Constructsa
KLP-15
construct
KLP-15239-587
KLP-15215-587
KLP-15200-587
KLP-15185-587
KLP-15148-587
predicted no.
of amino acids
in coiled coil
7
31
46
61
97
N-terminal
sequencing
calculated
molecular
massb(Da)
40 408
42 481
45 097
46 752
50 864
measured
molecular
massc(Da)
40 479
42 416
45 143
46 819
50 839
estimated
molecular
mass (kDa)d
association
statee
monomer
monomer + dimer
monomer + dimer
monomer + dimer
dimer
*GSSHHHHHHSSGLVPRGSHMASDVV
*ASHHHHHHPWASYRV
ndf
ndf
ndf
44/97
75
88
94
aThe initial methionine residue is missing due to N-terminal excision in E. coli (49) as shown by N-terminal sequencing. Klp15 constructs
cloned into pET17 start with an Ala, whereas constructs cloned into pET28 start with a Gly.bCalculated from the primary sequences. The additional
N-terminal residues, including the linker, the His tag (ASHHHHHHPWAS or GSSHHHHHHSSGLVPRGSHMAS), and missing methionine residues,
are taken into account.cDetermined by MALDI-TOF.dEstimation of molar mass determined by FPLC gel filtration on a Superose 12 column.
eFrom analytical ultracentrifugation sedimentation velocity experiments.fNot determined.
6530 Biochemistry, Vol. 44, No. 17, 2005
Robin et al.