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Title: Mammalian Kinesin-3 Motors Are Dimeric In Vivo and Move by Processive Motility upon Release of Autoinhibition}
Article:
Abstract:
Kinesin-3 motors drive the transport of synaptic vesicles and other membrane-bound organelles in neuronal cells. In the absence of cargo, kinesin motors are kept inactive to prevent motility and ATP hydrolysis. Current models state that the Kinesin-3 motor KIF1A is monomeric in the inactive state and that activation results from concentration-driven dimerization on the cargo membrane. To test this model, we have examined the activity and dimerization state of KIF1A. Unexpectedly, we found that both native and expressed proteins are dimeric in the inactive state. Thus, KIF1A motors are not activated by cargo-induced dimerization. Rather, we show that KIF1A motors are autoinhibited by two distinct inhibitory mechanisms, suggesting a simple model for activation of dimeric KIF1A motors by cargo binding. Successive truncations result in monomeric and dimeric motors that can undergo one-dimensional diffusion along the microtubule lattice. However, only dimeric motors undergo ATP-dependent processive motility. Thus, KIF1A may be uniquely suited to use both diffuse and processive motility to drive long-distance transport in neuronal cells.
Introduction:
Kinesin motors drive the long-distance transport of membrane-bound cargoes along microtubules. Long-distance transport is particularly important in neuronal cells whose length and polarity require robust sorting and transport of cargoes to pre- and postsynaptic destinations. Transport of synaptic vesicle precursors to axon terminals is driven by members of the Kinesin-3 family, the mammalian KIF1A, and Caenorhabditis elegans Unc104 motors[1]. Loss of KIF1A or Unc104 function results in decreased synaptic vesicles in axonal growth cones, and early death[1]. Thus, understanding how kinesin motors are regulated to enable transport of the correct cargo to the proper cellular destination at the relevant time is an important biological problem. In the absence of cargo, kinesin motors are kept inactive to prevent futile ATP( adenosine triphosphate) hydrolysis and motility. Two models have been proposed for how activity is suppressed in the absence of cargo. The first model posits that dimeric motors are regulated by an autoinhibitory mechanism. Autoinhibition typically involves a folded state that enables the motor's own tail domain to interact with and inhibit its motor domain. This model is based on a large body of work on the Kinesin-1 motor( formerly conventional kinesin or KIF5)[2–5]. In recent years, this model has received increasing experimental support from studies on kinesin motors involved in diverse functions such as epithelial polarity, intraflagellar transport, and mitosis[6–8]. Interestingly, autoinhibition may be a general model for motor regulation, as two well-studied members of the myosin family, nonmuscle myosin II and myosin V, exist in a folded inactive state[9–11]. Autoinhibition enables precise spatial and temporal regulation of motors and may be relieved by cargo binding[6, 12], phosphorylation[8], or other mechanisms. The second model states that motor activity is regulated by transition from a monomeric to dimeric state. Evidence for this model comes from studies on KIF1A/Unc104 motors where the full-length motors exist in a monomeric, inactive state[13–15]. Unc104 activity can be increased by forced dimerization or by an increase in the local concentration of the motor on liposomes[16–18]. Thus, cargo-induced dimerization would enable KIF1A/Unc104 motors to coordinate their two motor domains and step processively in a “hand-over-hand” fashion[19, 20]. The cargo-induced dimerization model has gained support from recent studies on the myosin family member myosin VI[21–24]. In this case, the cargo-binding tail domain exists in two mutually exclusive situations: either directly inhibiting the catalytic head in the monomeric state or mediating dimerization on the cargo membrane[25]. The cargo-induced dimerization model for Kinesin-3 motors is primarily based on work with recombinant CeUnc104 proteins. Whether mammalian KIF1A motors are regulated by cargo-induced dimerization has never been tested. In addition, although a region of the KIF1A stalk domain has been shown to inhibit microtubule binding of the motor domain[26], it is unclear how this potential autoinhibitory segment fits in the context of the two models for motor regulation. Furthermore, the sequences that facilitate dimerization remain to be identified. Here, we test the models for regulation of mammalian KIF1A motors and relate activity to the monomer/dimer state. We show that expressed and endogenous mammalian KIF1A motors exist in a dimeric, inactive state. Thus, cargo-induced dimerization is not a valid model for mammalian KIF1A motors. Rather, we provide support for an autoinhibition mechanism by identifying the sequences and mechanisms required for dimerization and inhibition. Finally, we show that only dimeric motors undergo processive, ATP-driven motility.
Discussion:
We provide four lines of experimental evidence( chemical crosslinking, coimmunoprecipitation, FRET, and photobleaching) to demonstrate that FL KIF1A expressed in mammalian cells exists as a dimeric protein. A concentration-dependent push to the dimeric state seems unlikely since crosslinking, coimmunoprecipitation, and photobleaching assays are carried out under dilute conditions. We also show that endogenous KIF1A behaves as a dimeric motor by crosslinking and sucrose gradient sedimentation. The sedimentation behavior of endogenous KIF1A was previously interpreted, based on comparisons to size standards, as indicating a protein of approximately 200 kDa and thus a monomeric molecule[13]. However, we show that the sedimentation behavior of endogenous KIF1A is consistent with a dimeric state when compared to Kinesin-1 and KIF1A motors whose oligomeric state has been confirmed in alternative assays. The sedimentation behavior of KIF1A is not surprising considering the behavior of other kinesin motors in these assays. Values of 6. 7 S have been reported for Kinesin-1( ∼340 kDa)[32], 8. 6–9. 8 S for the heterotrimeric Kinesin-2 motor KIF3A/KIF3B/KAP( ∼300 kDa)[33, 34], and 6. 8–7. 9 S for the homodimeric Kinesin-2 motor CeOSM-3( ∼160 kDa)[7, 34]. Thus, the oligomeric state of kinesin motors cannot be conclusively determined in sucrose gradients, and perhaps not in other biochemical analyses, by using MW standards and without considering other variables such as protein conformation. In conclusion, our data provide strong support for the idea[6, 35] that, like Kinesin-1 and Kinesin-2 motors, Kinesin-3 family members are dimeric motors. Our results further show that the NC plays an important role in mediating dimerization. This is consistent with studies showing coil formation by peptides corresponding to the predicted NC regions of MmKIF1A and CeUnc104[15, 30]. Importantly, we show that several residues in the hinge segment C-terminal to the NC are required for dimerization and processive motility, providing an explanation for why previous constructs that lack a full NC resulted in monomeric motors[16, 28, 31, 36, 37]. A role for the hinge region in dimerization and/or processive motility has been noted for other kinesins, most notably fungal Kinesin-1[38]. Our results demonstrate that KIF1A motors can exist in a dimeric but autoinhibited state. These results do not support a model for cargo-mediated dimerization of Kinesin-3 motors but rather suggest a simple model in which cargo binding relieves autoinhibition of dimeric motors. Using truncation mutants, we identified two regions that contribute to different mechanisms of autoinhibition. First, the FHA and CC2 domains inhibit the interaction of KIF1A with microtubules as KIF1A( 1–491) motors can bind to microtubules but cannot undergo processive motility, consistent with previous work[39]. Second, the CC1 domain regulates processive motility as KIF1A( 1–393) motors can both bind to microtubules and undergo processive motility. We show that CC1 blocks processive motility by interference with the formation of dimeric motors, perhaps due to an intramolecular interaction with the NC as seen in truncated CeUnc104 motors[28]. This interaction may contribute to the altered mobility of( 1–491) motors upon crosslinking. Further studies are required to test whether CC1-mediated inhibition occurs in the FL motor. In this respect, the interaction of CC1 and NC could serve to keep the two motor domains separated and/or uncoordinated, similar to the role of kinesin light chain( KLC) in the autoinhibition of Kinesin-1[2]. As two separate mechanisms are also involved in the autoinhibition of Kinesin-1[2], the dual-inhibition mode may be a common way to inhibit the activity of kinesin motors in the absence of cargoes. Autoinhibition is a common regulatory strategy used in diverse biological systems[40]. In terms of motor regulation, autoinhibition prevents futile ATP hydrolysis and allows rapid and specific control of motor activity both temporally and spatially. Autoinhibition has now been shown to regulate members of the Kinesin-1, Kinesin-2, and Kinesin-3 families( this study and[5–7]). Thus, the evolutionary development of new kinesin families by the attachment of novel cargo-binding domains onto the catalytic kinesin core may have simultaneously ensured that each family adopt a unique mechanism for autoinhibition. Our finding that endogenous KIF1A motors exist in a dimeric state indicates that the physiological form that drives processive motion of cargoes in cells is likely to be the dimeric motor. We show that the motility of dimeric KIF1A motors is characterized by smooth, unidirectional movement along microtubules and occurs with an average velocity comparable to vesicles moving in vivo[39, 41]. Whether dimeric KIF1A motors utilize a hand-over-hand mechanism like Kinesin-1 is a question for future experiments. That both monomeric and dimeric forms of KIF1A undergo 1D diffusion on microtubules likely indicates a nonspecific binding to the microtubule that enhances processivity. The ability of dimeric KIF1A motors to undergo both diffusive motion and processive motility is unique among kinesins involved in vesicle transport. Yet, it is interesting that 1D diffusion along microtubules has been reported for all three motor classes. The homotetrameric Kinesin-5 motor Eg5 undergoes ATP-independent 1D diffusion in the absence of cargo and switches to ATP-dependent processive motility in the presence of cargo, resulting in the sliding antiparallel microtubules during formation of the mitotic spindle[42, 43]. Similarly, mitotic centromere-associated kinesin( MCAK), a Kinesin-13 motor, uses ATP-independent 1D diffusion to reach microtubule plus ends where ATP-dependent catalytic activity results in depolymerization of microtubules[44]. Surprisingly, Myosin Va can undergo rapid diffusion along the microtubule lattice in vitro[45]. Finally, diffusive motion of the dynactin complex along microtubules may facilitate cytoplasmic dynein processivity[46]. KIF1A may thus be uniquely positioned among vesicular motors to drive long-distance motility using 1D diffusive motion to tether motors to microtubules, as well as ATP hydrolysis for force production.
Molecular motors transport a wide variety of cellular cargoes that are important for diverse cellular phenomena such as mitosis , polarity , motility , and secretion . Motor activity must be tightly regulated to ensure that ATP hydrolysis and processive motility occur only upon coupling to the correct cargo . In neuronal cells , Kinesin-3 motors drive the transport of presynaptic vesicles and other membrane-bound organelles along microtubule tracks . Yet the mechanisms of Kinesin-3 motor activation and motility remain controversial . In this study , we examine the regulation and motile properties of the Kinesin-3 motor KIF1A . We show that in the absence of cargo , KIF1A motors exist in a dimeric inactive state that is maintained by two distinct autoinhibitory mechanisms . This suggests a simple model for activation of dimeric motors upon cargo binding . We also show that dimeric motors can undergo two mechanisms of motility along microtubule tracks: one-dimensional diffusion and ATP-driven processive motility . This unique property may facilitate the ability of KIF1A to drive long-distance vesicular transport in neuronal cells .