(Received for publication, September 13, 1995; and in revised form, November 30, 1995 )
From the e 42, 80336 Munich,
Federal Republic of Germany
Neurospora kinesin (Nkin) is a distant relative of the
family of conventional kinesins, members of which have been identified
in various animal species. As in its animal counterparts, Nkin most
likely is an organelle motor. Because it is a functional homologue of
the kinesin heavy chain of higher eukaryotes, its biophysical and
motility properties were compared with those of other conventional
kinesins. Purified Nkin behaves as a homodimeric complex composed of
two subunits of a 105-kDa polypeptide. Based on its hydrodynamic
properties (Stokes radius and sedimentation coefficient), Nkin is an
elongated molecule, although it is more compact than its animal
counterparts. A detailed comparison of the motility properties of Nkin
with those of animal conventional kinesins reveals similarities and
some intriguing differences. Nkin is less effective than other kinesins
in the use of natural nucleoside triphosphates but responds to a
selection of ATP analogues in a similar fashion as mammalian kinesin.
Even in the presence of saturating concentrations of ATP, Nkin is
significantly more sensitive to ADP or tripolyphosphate than other
kinesins. Both the ATP-driven microtubule gliding activity and the
microtubule-stimulated ATPase activity of Nkin obey Michaelis-Menten
kinetics. Surprisingly, however, the K values for both these activities are approximately an order
of magnitude higher than those of other kinesins. Whether the low
affinity for ATP suggested by these high K
values is related to the high rate of motility remains to be
determined.
Kinesin and kinesin-like proteins comprise a superfamily of
microtubule-dependent molecular motors important for various functions
in intracellular organelle movements, mitosis, meiosis, and karyogamy
(for reviews see Goldstein(1993), Walker and Sheets(1993), and Bloom
and Endow(1994)). Among the at least seven families and several (as
yet) single representatives, the family of ``conventional''
kinesins is the best characterized. The founding member of this family
was identified concurrently in chick brain (Brady, 1985) and squid
neural tissue (Vale et al., 1985a). Motors closely related to
squid kinesin have subsequently been isolated from various tissues and
organisms including bovine brain (Bloom et al., 1988;
Kuznetsov and Gelfand, 1986), sea urchin eggs (Scholey et al.,
1985), Drosophila (Saxton et al., 1988), and bovine
adrenal medulla (Murofushi et al., 1988). A gene closely
related to conventional kinesin has been found in Caenorhabditis
elegans (Patel et al., 1993). In addition, recent
evidence suggests that conventional kinesin may exist in two or more
isoforms, at least in mammals (Navone et al., 1992; Niclas et al., 1994). Conventional kinesins have several properties
in common: (i) they can be purified from cell extracts in sufficient
quantities for biochemical and biophysical studies (Vale et
al., 1985a; Brady, 1985; Scholey et al., 1985); (ii) they
are composed of two 110-130-kDa heavy chains and two
55-85-kDa light chains (Bloom et al., 1988; Kuznetsov et al., 1988); (iii) the heavy chains possess a globular
N-terminal motor domain where the ATP and microtubule binding sites are
located, a central -helical (coiled-coil) stalk, and a globular
C-terminal domain where the light chains bind (Hirokawa et
al., 1989; Yang et al., 1989; Gauger and Goldstein,
1993); (iv) both the heavy and light chains exhibit a high degree of
sequence homology throughout their length; (v) finally, the members of
this family have been strongly implicated as force-generating proteins
for the transport of membrane-bound organelles (Dabora and
Sheetz(1988), Schroer et al.(1988), and Pfister et
al.(1989); for review see Walker and Sheetz (1993) and Bloom and
Endow(1994)). Taken together these properties suggest that the
conventional kinesins are a family of closely related proteins whose
molecular and biochemical properties are remarkably highly conserved.
Recently, we have identified an organelle motor from a
``lower'' eukaryote, the filamentous fungus Neurospora
crassa. A molecular phylogenetic analysis using the N-terminal
motor domain suggests that this motor, termed Nkin, ()is a
distant relative of the family of conventional kinesins identified in
animals. Nkin exhibits several unusual structural and functional
properties, such as a high rate of microtubule transport, a lack of
copurifying polypeptides, a second p-loop motif, and the presence of a
highly conserved peptide motif in the C terminus. The first organelle
motor related to conventional kinesins identified in fungi, Nkin
represents an intriguing ``outsider'' of this otherwise
homogeneous protein family. To link the biochemical and biophysical
properties of these motors to their biological function, we have
analyzed the enzymatic, biophysical, and motility properties of Nkin
and compared them, where possible, with those of animal kinesins.
Native Nkin was isolated from cytoplasmic extracts of hyphae
based on its nucleotide-sensitive binding to and release from pig brain
microtubules (Steinberg and Schliwa, 1995). A typical experiment
yielded 10-20 µg of motor from 30 g of packed hyphae. We
routinely used apyrase to deplete ATP from cytoplasmic extracts for
microtubule binding, but motor could also be isolated using AMPPNP as
originally described (Brady, 1985; Vale et al., 1985a). The
best purification was achieved by the combination of apyrase and a low
amount (0.2 mM) of AMPPNP, followed by gel filtration over a
superose-6 fast protein liquid chromatography column, which yielded
protein fractions that are 85-90% pure Nkin by densitomety of
Coomassie-stained polyacrylamide gels (Fig. 1). In the absence
of ATP and microtubules, the motor quickly lost its ability to promote
microtubule motility. Therefore, care was taken to maintain a minimum
concentration of 0.05 mM ATP or ATPS during density
gradient centrifugation or gel filtration. As discussed in Steinberg
and Schliwa(1995) purified Nkin consists of a polypeptide doublet at
105/108 kDa, where the higher molecular mass form most likely
represents a phosporylated isoform whose proportion varied from
preparation to preparation. Despite our efforts, co-purifying
polypeptides could not be detected. Most of the experiments to be
described here were carried out with Nkin purified by gel filtration
and sucrose density gradient centrifugation. Some of the standard
microtubule gliding assays were also performed with material released
from microtubules with ATP, in which Nkin constitutes about 30% of the
total protein. No differences were found in these experiments to those
obtained with gel-filtered material.
Figure 1: Isolation of Neurospora kinesin. An organelle-free cytoplasmic extract of Neurospora wild type cell (S2) was incubated for 1 h at 4 °C with 0.8 mg/ml taxol-stabilized microtubules under ATP-depleting conditions and sedimented (P3). The pellet was resuspended in isolation buffer with 50 mM salt and centrifuged through a 10% sucrose cushion. The supernatant (S4) was discarded, and the microtubule pellet (P4) was resuspended in release buffer containing 10 mM MgATP. For further purification the kinesin-containing supernatant (S5) was run over a gel filtration column and highly purified Neurospora kinesin was obtained (NKIN). Microtubule gliding was observed in P3, P4, S5, and the column fraction. The proteins were analyzed in SDS-polyacrylamide gel electrophoresis on a 7% gel and stained with Coomassie Brilliant Blue.
Figure 2:
Stokes radius of Neurospora kinesin. A series of globular proteins were separated by gel
filtration and V/V
was
plotted against the Stokes radii of these calibration proteins (n = 3; Andrews(1970)). The average V
/V
of the Neurospora motor (n = 9) was determined, and its Stokes
radius was calculated from the regression equation. BSA,
bovine serum albumin;
-gal,
-galactosidase.
Figure 3:
Sedimentation coefficient of Neurospora kinesin. Globular calibration proteins were
separated on a 4.5-ml 5-20% sucrose density gradient (n = 2). Fractions of 70 µl each were analyzed by
SDS-polyacrylamide gel electrophoresis, and the elution volume for each
protein was plotted against its sedimentation coefficient. The mean
elution volume (n = 5) was used to calculate the
sedimentation coefficient of Nkin from the regression equation. BSA, bovine serum albumin.
The fundamental hydrodynamic and diffusion properties of Nkin reported here are those of an elongated molecule. Its Stokes radius, however, is considerably smaller than that of bovine kinesin, suggesting that Nkin is more compact than other conventional kinesins. This is reflected also in a smaller axial ratio of 12:1, as opposed to bovine brain kinesin, whose axial ratio is 20:1 (Bloom et al., 1988) for the ``folded'' form of kinesin and more like 40:1 for the fully extended molecule (Hackney et al., 1992).
It was reported recently that microtubules polymerized in the presence of a nonhydrolyzable GTP analogue, GMPCPP, have a higher flexural rigidity (appear stiffer; Venier et al., 1994) and that they are transported at a 30% higher rate in gliding assays than GTP microtubules (Vale et al., 1994). When gliding assays were done with GMPCPP microtubules, such an increase in the rate of microtubule movement was not observed, suggesting that microtubule stiffness does not alter microtubule velocity in the case of Nkin.
MgATP-dependent microtubule gliding has been found to be inhibited
by a variety of compounds. In contrast to other conventional kinesins,
Nkin appeared to be much more sensitive to some of these inhibitors.
Thus, MgADP, a competitive inhibitor (Cohn et al., 1989),
reduced the rate of microtubule gliding to about 50% at a ratio of
1:1 to MgATP, whereas a 4-fold excess was needed for sea urchin
kinesin (Cohn et al., 1989). Nkin also was more sensitive
toward AMPPNP. Both findings are consistent with a lower affinity of
Nkin for ATP, reflected also in the K
values of
both ATPase activity and microtubule motility (see below).
Microtubule gliding activity of Nkin was relatively insensitive to sodium orthovanadate and N-etylmaleimide. The mode of inhibition is unexpected. With increasing concentrations of both reagents, there was a progressive release of microtubules from the coverslip surface, whereas the rate of movement of microtubules seemed virtually unaffected. At concentrations of orthovanadate of >70 µM and concentrations of N-etylmaleimide of >3-5 mM, virtually no microtubules were left attached to the coverslip. However, a microtubule occasionally contacting the surface may be transported for several micrometers at normal rates before detaching again.
ATP produced microtubule gliding in a
saturable manner. The velocity obeyed a generalized
Michaelis-Menten-like law (Leibler and Huse, 1993), as demonstrated by
linearity in double-reciprocal plots of motility rates versus ATP concentration (Lineweaver-Burk plot; Fig. 4). In
independent experiments with three different preparations of Nkin,
linear regression analysis yielded apparent K values of 187, 340, and 393 µM ATP.
Figure 4:
Relationship between microtubule gliding
velocity and ATP concentration. Column-purified motor was attached to
clean coverslips, 0.06 mg/ml taxol-stabilized microtubules were added,
and the probe was sealed in a flow-through chamber. After rinsing the
chamber with ATP-free buffer the desired MgATP concentration was added,
and motility was recorded on a video tape. The measured velocities were
plotted against the ATP concentration (top). The K was calculated from a Lineweaver-Burk
plot (bottom).
Native Nkin
possessed an ATPase activity that was stimulated approximately 6-fold
by microtubules (Steinberg and Schliwa, 1995). As already discussed by
Huang and Hackney(1994) and Steinberg and Schliwa (1995), the ATPase
rates determined in these assays are inconsistent with the rates of
motility observed, suggesting that the ATPase properties of kinesin
isolated by conventional procedures do not reflect the properties of
the molecule in vivo. Like microtubule motility, the ATPase
activity obeyed Michaelis-Menten kinetics (Fig. 5), yielding K values of 112, 145, and 194 µM ATP
in three indepentent experiments. The ratio of the K
for motility (average 150 µM) and the K
for ATPase activity (average 306
µM) was
2
(Leibler and Huse, 1993). Studies
with sea urchin, Drosophila, and bovine brain kinesin have
yielded K
values for microtubule gliding ranging
from 10 to 60 µM (Porter et al., 1987; Saxton et al., 1988; Cohn et al., 1989; Howard et
al., 1989); K
values of ATPase activity have
been determined to be 10-20 µM for bovine brain
kinesin (Kuznetsov et al., 1986, 1989) and 31 µM for recombinant Drosophila kinesin (Gilbert and Johnson,
1993). Thus for both K
values, the values of Nkin
are approximately an order of magnitude higher. Whether the high rate
of microtubule movement is related to the low affinity for ATP remains
to be determined. In a theoretical analysis, Leibler and Huse(1993)
compared the motors myosin and kinesin and predicted the ratio of the K
for motility and the K
for
ATPase activity to be approximately 10 for myosin and 1-2 for
kinesin. Even though the K
values of Nkin differ
markedly from those of other kinesins, the ratio of the Michaelis-like
constants for velocity and ATPase activity is approximately 2, as in
the case of other conventional kinesins. Thus the basic features of the
cross-bridge cycle of Nkin probably are similar to those of other
kinesins. Nkin therefore seems to be a molecular motor of the
``porter'' type (Leibler and Huse, 1993), in contrast to
myosin and probably also dynein, which belong to the
``rower'' class of molecular motors with a ratio of the K
values of >10. The distinction between these
two classes of motors on the basis of their K
values for motility and ATPase activity is in agreement with
recent studies that directly demonstrate that the chemomechanical
cycles of kinesin and myosin are indeed fundamentally different
(Romberg and Vale, 1993; Gilbert et al., 1995). It will be
interesting to determine whether the high rate of motility of Nkin is
based on a larger step size, a shorter cycle time, or both.
Figure 5:
Microtubule-stimulated ATPase activity of Neurospora kinesin. The ATPase activity was determined by
measuring the change in the optical density at 640 nm caused by the
complex of phosphomolybdate and Malachite Green (Itaya and Ui, 1965).
The enzymatic activity was monitored in the presence of 1 mg/ml taxol
stabilized microtubules and plotted against the ATP concentration (top). The K was calculated from
a Lineweaver-Burk plot (bottom).