Kinesin-2 KIF3AB Exhibits Novel ATPase Characteristics

Albracht, Clayton D.; Rank, Katherine C.; Obrzut, Steven; Rayment, Ivan; Gilbert, Susan P.

doi:10.1074/jbc.m114.583914

Journal of Biological Chemistry

2014

DOI: 10.1074/jbc.m114.583914

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Kinesin-2 KIF3AB Exhibits Novel ATPase Characteristics

Clayton D. Albracht

Katherine C. Rank

Steven Obrzut

et al.

Abstract: Background: KIF3AB is a heterotrimeric plus-end-directed kinesin-2 motor, implicated in intraflagellar transport. Results: KIF3AB shows rate-limiting ADP release upon microtubule collision and an unusual apparent weak ATP affinity observed at microtubule⅐KIF3AB dissociation. Conclusion:The presteady-state kinetics suggest a novel ATPase mechanism for KIF3AB stepping. Significance: The KIF3AB kinetics reveal the mechanistic diversity that kinesin motors exhibit for cargo transport.

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Cited by 15 publications

(44 citation statements)

References 64 publications

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“…Hence, there is no evidence that discrepancies between the two studies result from differences in the motor constructs employed. As argued below, we contend that our results are in fact consistent with the experimental data of Albracht et al (64), but we differ significantly on the interpretation of the results.…”

supporting

confidence: 78%

“…The first conclusion from the Albracht et al study (64) was that ADP release at 12 s Ϫ1 is the rate-limiting step in the KIF3A/B hydrolysis cycle. This conclusion came from a sequential release experiment (mADP-loaded motors are flushed against microtubules in the presence of saturating ATP, leading to a fall in fluorescence) that was very similar to our sequential release experiments in Fig.…”

mentioning

confidence: 99%

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Processivity of the Kinesin-2 KIF3A Results from Rear Head Gating and Not Front Head Gating

Chen

Arginteanu

Hancock

2015

Journal of Biological Chemistry

115

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Background: KIF3A/B is less processive than kinesin-1, particularly under load. Results: In an engineered KIF3A dimer, intramolecular strain in the two-head-bound state does not alter nucleotide binding. In ADP, the motor dissociates slowly from microtubules. Conclusion: KIF3A lacks front head gating and maintains processivity through weak microtubule binding. Significance: During bidirectional transport, kinesin-2 is expected to detach more readily than kinesin-1.

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supporting

confidence: 78%

mentioning

confidence: 99%

Processivity of the Kinesin-2 KIF3A Results from Rear Head Gating and Not Front Head Gating

Chen

Arginteanu

Hancock

2015

Journal of Biological Chemistry

115

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“…KIF3 Protein Expression and Purification-KIF3 motors were expressed in Escherichia coli BL21-CodonPlus (DE3)-RIL cell line (Stratagene, La Jolla, CA) for purification as described in detail previously (30,32). To achieve the purification of KIF3AC heterodimers, sequential affinity columns were used.…”

mentioning

confidence: 99%

“…Pulse-Chase Kinetics of ATP Binding-To determine the time course of ATP binding, a chemical quench-flow instrument (RQF-3, KinTek Corp.) was used (32,33). The MT⅐KIF3AC complex (5 M KIF3AC/40 M MTs or 10 M KIF3AC/40 M MTs, syringe concentrations) was rapidly mixed with MgATP plus radiolabeled [␣-32 P]ATP and 300 mM KCl (syringe concentrations) for times ranging from 5 to 200 ms in the instrument.…”

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confidence: 99%

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Fast or Slow, Either Head Can Start the Processive Run of Kinesin-2 KIF3AC

Zhang

Rayment

Gilbert

2016

Journal of Biological Chemistry

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Mammalian KIF3AC contains two distinct motor polypeptides and is best known for its role in organelle transport in neurons. Our recent studies showed that KIF3AC is as processive as conventional kinesin-1, suggesting that their ATPase mechanochemistry may be similar. However, the presence of two different motor polypeptides in KIF3AC implies that there must be a cellular advantage for the KIF3AC heterodimer. The hypothesis tested was whether there is an intrinsic bias within KIF3AC such that either KIF3A or KIF3C initiates the processive run. To pursue these experiments, a mechanistic approach was used to compare the pre-steady-state kinetics of KIF3AC to the kinetics of homodimeric KIF3AA and KIF3CC. Kinesin-2 subfamily members are best known for their roles in long distance cargo transport, but unlike other processive kinesins, the kinesin-2 subfamily includes both homodimeric and heterodimeric kinesins (reviewed in Refs. 1-3). In mammals, there are four kinesin-2 subfamily genes: KIF3A, KIF3B, KIF3C,. When expressed, KIF17 forms homodimers, yet KIF3A associates with either KIF3B or KIF3C to form the heterodimers KIF3AB and KIF3AC. In each case, these kinesin-2 molecular motors associate with nonmotor adaptor proteins to link the kinesin to its specific cargo (6, 15, 18 -27). Of the kinesin-2 adaptors, the kinesin-associated protein KAP with KIF3AB has resulted in the designation of KIF3AB-KAP as a heterotrimeric kinesin (6, 18 -22, 26). KIF3AB and KIF17 have been widely studied in multiple model organisms including Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, as well as Mus musculus to understand their roles in intraflagellar transport and organelle transport for many other cellular functions (1-3). In contrast, KIF3AC has not been as well studied because there is not a true KIF3C ortholog in many of these model organisms. KIF3C exhibits a signature motif conserved in mammalian species, a 25-residue insert in loop L11 of the catalytic motor domain, which is enriched in glycines and serines (7, 28 -30). This L11 insert is not present in other kinesins. Furthermore, even though KIF3AB and KIF3AC share similarities in protein sequence and structure, KIF3AC appears to function specifically in neurons rather than ubiquitously as do KIF3AB and KIF17 (1, 2, 28).Recent single molecule studies revealed that heterodimers of KIF3AC and KIF3AB were as processive as kinesin-1 K560 based on their run lengths, indicating that KIF3AC and KIF3AB have the capacity for long distance intracellular transport (30). Furthermore, to begin to tease apart the contributions of each motor head, homodimers of KIF3AA, KIF3BB, and KIF3CC were generated and characterized. The results for KIF3AA in comparison with KIF3CC were striking because despite sequence similarity between KIF3A and KIF3C, KIF3AA was a very fast and processive kinesin, yet KIF3CC, although processive, was exceedingly slow (Table 1). In contrast, the properties of KIF3AA and KIF3BB were more similar to each other (30,31). This obvious disparity between...

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Kinesin-2 KIF3AC and KIF3AB Can Drive Long-Range Transport along Microtubules

Guzik-Lendrum

Rank

Bensel

et al. 2015

Biophysical Journal

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Mammalian KIF3AC is classified as a heterotrimeric kinesin-2 that is best known for organelle transport in neurons, yet in vitro studies to characterize its single molecule behavior are lacking. The results presented show that a KIF3AC motor that includes the native helix α7 sequence for coiled-coil formation is highly processive with run lengths of ∼1.23 μm and matching those exhibited by conventional kinesin-1. This result was unexpected because KIF3AC exhibits the canonical kinesin-2 neck-linker sequence that has been reported to be responsible for shorter run lengths observed for another heterotrimeric kinesin-2, KIF3AB. However, KIF3AB with its native neck linker and helix α7 is also highly processive with run lengths of ∼1.62 μm and exceeding those of KIF3AC and kinesin-1. Loop L11, a component of the microtubule-motor interface and implicated in activating ADP release upon microtubule collision, is significantly extended in KIF3C as compared with other kinesins. A KIF3AC encoding a truncation in KIF3C loop L11 (KIF3ACΔL11) exhibited longer run lengths at ∼1.55 μm than wild-type KIF3AC and were more similar to KIF3AB run lengths, suggesting that L11 also contributes to tuning motor processivity. The steady-state ATPase results show that shortening L11 does not alter kcat, consistent with the observation that single molecule velocities are not affected by this truncation. However, shortening loop L11 of KIF3C significantly increases the microtubule affinity of KIF3ACΔL11, revealing another structural and mechanistic property that can modulate processivity. The results presented provide new, to our knowledge, insights to understand structure-function relationships governing processivity and a better understanding of the potential of KIF3AC for long-distance transport in neurons.

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Kinesin-2 KIF3AB Exhibits Novel ATPase Characteristics

Cited by 15 publications

References 64 publications

Processivity of the Kinesin-2 KIF3A Results from Rear Head Gating and Not Front Head Gating

Processivity of the Kinesin-2 KIF3A Results from Rear Head Gating and Not Front Head Gating

Fast or Slow, Either Head Can Start the Processive Run of Kinesin-2 KIF3AC

Kinesin-2 KIF3AC and KIF3AB Can Drive Long-Range Transport along Microtubules

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