KIF1D is a fast non-processive kinesin that demonstrates novel K-loop-dependent mechanochemistry - PubMed (original) (raw)
KIF1D is a fast non-processive kinesin that demonstrates novel K-loop-dependent mechanochemistry
K R Rogers et al. EMBO J. 2001.
Abstract
The KIF1 subfamily members are monomeric and contain a number of amino acid inserts in surface loops. A particularly striking insertion of several lysine/arginine residues occurs in L12 and is called the K-loop. Two recent studies have employed both kinetic and single-molecule methods to investigate KIF1 motor properties and have produced very different conclusions about how these motors generate motility. Here we show that a hitherto unstudied member of this group, KIF1D, is not chemically processive and drives fast motility despite demonstrating a slow ATPase. The K-loop of KIF1D was analysed by deletion and insertion mutagenesis coupled with characterization by steady state and transient kinetics. Together, the results indicate that the K-loop not only increases the affinity of the motor for the MT, but crucially also inhibits its subsequent isomerization from weak to strong binding, with coupled ADP release. By stabilizing the weak binding, the K-loop establishes a pool of motors primed to undergo their power stroke.
Figures
Fig. 1. Kinetic properties of KIF1D. (A) Effect of salt concentration on KIF1D MT-activated ATPase. Assays in (A)–(C) were carried out in 25 mM PIPES, 1 mM MgCl2, 1 mM DTT pH 6.9 with the addition of NaCl to the specified concentration at 22°C. The motor concentration was 0.1 µM. Each data set was fitted to a rectangular hyperbola to give a maximum ATPase rate (_K_cat) and the MT concentration required for half-maximal activation [K(0.5)MT]. Open circles, 100 mM NaCl; squares, 50 mM NaCl; diamonds, 25 mM NaCl; filled circles, no NaCl. (B) Comparison of the MT-activated ATPase of KIF1D and single-headed kinesin (rkin340GFP). Open circles, rkin340GFP; filled squares, KIF1D. (C) Representative stop-flow trace of KIF1D. 0.5 µM KIF1D was labelled with 2 µM mantATP and rapidly mixed with MT (20 µM). Concentrations are after mixing in the stop-flow chamber. Solid line is the best fit to a single exponential. (D) MT-activated mantADP release from the KIF1D motor. Each point is the average of three traces. Solid line shows the best fit to a rectangular hyperbola and gives the maximum mantADP release rate K(mantADP) of 21.35 s–1 and the MT concentration required for half-maximal activation K(0.5mantADP) of 0.18 µM.
Fig. 2. Histogram of MT motility of KIF1D in multiple-motor assays. Assays were carried out in 25 mM PIPES, 25 mM NaCl, 1 mM MgCl2, 1 mM DTT, 1 mM ATP, 0.1 mg/ml casein pH 6.9. Mean velocity is 2.01 ± 0.21 µM/s. Bin size, 0.1 µm/s.
Fig. 3. Schematic representation of KIF1D and kinesin motor constructs. The wild-type L12 loop regions are shown in grey and the mutant L12 loop outlined in black. The numbers above each construct show the position of the first and last amino acids of the motor sequence. The L12 loop region is expanded to show the amino acid sequence of wild-type KIF1D and rat kinesin (rkin) together with the KIF1D-K and rkin+K mutants. GST, glutathione _S_-transferase; GFP, green fluorescence protein; His, His6 affinity tag.
Fig. 4. Kinetic properties of the K-loop mutants. (A) Steady state ATPase rates of KIF1D and KIF1D-K motors. The MT-activated ATPase assays were performed as in Figure 1 in the low salt buffer. Circles, KIF1D; squares, KIF1D-K. (B) MT-activated mantADP release of KIF1D and KIF1D-K. Each point is the average of at least three traces. Experiments were performed as in Figure 1D. Open circles, KIF1D; squares, KIF1D-K. (C and D) MT-activated ATPase and MT-activated mantADP release of rkin340GFP and rkin340+KGFP motors, respectively. Circles, rkin340+KGFP; diamonds, rkin340GFP.
Fig. 5. rkin430+KGFP mantADP release kinetics and motility. (A and B) Representative fluorimeter traces of rkin430+KGFP. The motor was labelled with mantATP and rapidly mixed with: (A) MT (10 µM) with chase 1 mM ATP nucleotide; (B) MT (10 µM) alone. In (C) the motor was labelled with mantATP mixed with MTs to form a motor–MT complex. The release of the mantADP was monitored after rapid mixing with 1 mM ATP. The solid lines show the best fit to single exponentials. Assay conditions were as in Figure 3B. (D) Histogram of MT velocity supported by rkin430GFP (white bars) and rkin430+KGFP (grey bars). Bin size is 0.2 µM/s. The mean velocities of rkin430+KGFP and rkin430GFP are 139 ± 0.019 and 295 nm/s ± 0.032, respectively. Assays were carried out in BRB80 supplemented with 1 mM DTT, 0.5 mg/ml casein and 1 mM ATP.
Fig. 6. Flash photolysis measurement of the decrease in light scattering of the motor–MT complex after release of ATP. (A) KIF1D; (B) KIF1D-K; (C) rkin340GFP; and (D) rkin340+KGFP. Upper traces show the light scattering versus time of the motor–MT complex (i.e. dissociation) after the release of three different concentrations of ATP by three consecutive flashes of laser light. The lower panels show the rate constants from single exponential fits of each trace plotted against the concentration of released ATP.
Fig. 7. A two-step binding model for KIF1D–MT interaction. The motor binds the MT in two steps; first, the weakly bound or diffusional state followed by the strongly bound state. Critically the rate of isomerization into the strongly bound state is slow and therefore increases the length of time the motor spends in the M–ADP weakly bound state. The rate constant for isomerization from weakly bound to strongly bound M–ADP is estimated based on the MT-activated mantADP release rate of KIF1D; we argue that the isomerization is rate limiting in this assay. The release of mantADP from the strongly bound motor is assumed to be ∼298 s–1 from MT-activated mantADP release rates from KIF1D-K. Other steps are assumed to be fast, except phosphate release, which may set a limit on dissociation.
Fig. 8. Model of KIF1 cargo transport. We propose that the monomeric motors work in small teams (or arrays) on the cargo that is transported (e.g. a mitochondria). A large proportion of the motors in the team are in the weakly bound state (tilted, slack tether), with the K-loop tethering the cargo to the MT and inhibiting the transition to the strongly bound state. They do not exert significant drag on forward motion. Once strongly bound the motors undergo a power stroke to produce forward motion after which they revert to the weakly bound K-loop-tethered diffusional state. To maintain further forward motion, other weakly bound motors are recruited to undergo their strong interaction and consequent power stroke. If traction is lost the weakly bound motor maintains close association with the MT until other heads are recruited, thus maintaining cargo transport over longer distances.
References
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