Mammalian Kinesin-3 Motors Are Dimeric In Vivo and Move by Processive Motility upon Release of Autoinhibition

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.     

The CC1-FHA Tandem as a Central Hub for Controlling the Dimerization and Activation of Kinesin-3 KIF1A

Kinesin-3 KIF1A plays prominent roles in axonal transport and synaptogenesis. KIF1A adopts?a monomeric form in?vitro but acts as a processive dimer in?vivo. The mechanism underlying the motor dimerization is poorly understood. Here, we find that the CC1-FHA tandem of KIF1A exists as a stable dimer. The structure of CC1-FHA reveals that the linker between CC1 and FHA unexpectedly forms a β-finger hairpin, which integrates CC1 with FHA assembling a CC1-FHA homodimer. More importantly, dissociation of the CC1-FHA dimer unleashes CC1 and the β-finger, which are both essential for the motor inhibition. Thus, dimerization of the CC1-FHA tandem not only promotes the KIF1A dimer formation but also may trigger the motor activity via sequestering the CC1/β-finger region. The CC1-FHA tandem likely functions as a hub for controlling the dimerization and activation of KIF1A, which may represent?a new paradigm for the kinesin regulation shared by other kinesin-3 motors.     

KIF1D is a fast non-processive kinesin that demonstrates novel K-loop-dependent mechanochemistry

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.     

An intramolecular interaction between the FHA domain and a coiled coil negatively regulates the kinesin motor KIF1A

Motor proteins not actively involved in transporting cargoes should remain inactive at sites of cargo loading to save energy and remain available for loading. KIF1A/Unc104 is a monomeric kinesin known to dimerize into a processive motor at high protein concentrations. However, the molecular mechanisms underlying monomer stabilization and monomer-to-dimer transition are not well understood. Here, we report an intramolecular interaction in KIF1A between the forkhead-associated (FHA) domain and a coiled-coil domain (CC2) immediately following the FHA domain. Disrupting this interaction by point mutations in the FHA or CC2 domains leads to a dramatic accumulation of KIF1A in the periphery of living cultured neurons and an enhancement of the microtubule (MT) binding and self-multimerization of KIF1A. In addition, point mutations causing rigidity in the predicted flexible hinge disrupt the intramolecular FHA-CC2 interaction and increase MT binding and peripheral accumulation of KIF1A. These results suggest that the intramolecular FHA-CC2 interaction negatively regulates KIF1A activity by inhibiting MT binding and dimerization of KIF1A, and point to a novel role of the FHA domain in the regulation of kinesin motors.     

Monomeric and dimeric states exhibited by the kinesin-related motor protein KIF1A.

KIF1A, a kinesin-related motor protein that transports pre-synaptic vesicles in neurons, was originally presumed to translocate along microtubules (MT) as a monomer. Protein structure predictions from its amino acid sequence failed to identify the long coiled-coil domains typical of kinesins, which led researchers to believe it does not oligomerize into the canonical kinesin dimer. However, mounting evidence using recombinant chimeric protein indicates that KIF1A, like conventional kinesin, requires dimerization for fast, unidirectional processive movement along MTs. Because these studies are somewhat indirect, we wished to test the oligomerization state of native KIF1A, and to compare that to full-length recombinant protein. We have performed hydrodynamic analyses to determine the molecular weights of the respective complexes. Our results indicate that most native KIF1A is soluble and indeed monomeric, but recombinant KIF1A is a dimer. MT-binding studies also showed that native KIF1A did not bind to MTs in either the presence of AMP-PNP, apyrase, or adenosine triphosphate (ATP), but recombinant KIF1A bound to MTs most stably in the presence of ATP, indicating very different motor functional states. To further characterize KIF1A's dimerization potential, we prepared peptides corresponding to the neck domains of MmKIF1A and CeUnc104, and by circular dichroism spectroscopy compared these peptides for their ability to form coiled-coils. Interestingly, both MmKIF1A and CeUnc104 neck peptides formed homodimeric coiled-coils, with the MmKIF1A neck coiled-coil exhibiting the greater stability. Collectively, from our data and from previous studies, we predict that native KIF1A can exist as both an inactive monomer and an active homodimer formed in part through its neck coiled-coil domain.     

Defect in Synaptic Vesicle Precursor Transport and Neuronal Cell Death in KIF1A Motor Protein–deficient Mice

The nerve axon is a good model system for studying the molecular mechanism of organelle transport in cells. Recently, the new kinesin superfamily proteins (KIFs) have been identified as candidate motor proteins involved in organelle transport. Among them KIF1A, a murine homologue of unc-104 gene of Caenorhabditis elegans, is a unique monomeric neuron– specific microtubule plus end–directed motor and has been proposed as a transporter of synaptic vesicle precursors (Okada, Y., H. Yamazaki, Y. Sekine-Aizawa, and N. Hirokawa. 1995. Cell. 81:769–780). To elucidate the function of KIF1A in vivo, we disrupted the KIF1A gene in mice. KIF1A mutants died mostly within a day after birth showing motor and sensory disturbances. In the nervous systems of these mutants, the transport of synaptic vesicle precursors showed a specific and significant decrease. Consequently, synaptic vesicle density decreased dramatically, and clusters of clear small vesicles accumulated in the cell bodies. Furthermore, marked neuronal degeneration and death occurred both in KIF1A mutant mice and in cultures of mutant neurons. The neuronal death in cultures was blocked by coculture with wild-type neurons or exposure to a low concentration of glutamate. These results in cultures suggested that the mutant neurons might not sufficiently receive afferent stimulation, such as neuronal contacts or neurotransmission, resulting in cell death. Thus, our results demonstrate that KIF1A transports a synaptic vesicle precursor and that KIF1A-mediated axonal transport plays a critical role in viability, maintenance, and function of neurons, particularly mature neurons.     

15 A resolution model of the monomeric kinesin motor, KIF1A

A two-headed structure has been widely believed to be essential for the kinesin molecular motor to move processively on the track, microtubules. However, we have recently demonstrated that a monomeric motor domain construct of KIF1A (C351), a kinesin superfamily protein, moves processively, taking about 700 steps before being detached from microtubules. To elucidate the mechanism of its single-headed processivity, we examined the C351 -MT interaction by mutant analysis and high-resolution cryo-EM. Mutant analysis indicated the importance of a highly positively charged loop, the "K loop," for such processivity. A 15 A resolution structure unambiguously docked with the available atomic models revealed "K loop" as an extra microtubule-binding domain specific to KIF1A, and bound to the C terminus of tubulin. The site-specific cross-linking further confirmed this model.     

KIF1Bbeta2, capable of interacting with CHP,is localized to synaptic vesicles

Kinesin family proteins are microtubule-dependent molecular motors involved in the intracellular motile process. Using a Ca2+ -binding protein, CHP (calcineurin B homologous protein), as a bait for yeast two hybrid screening, we identified a novel kinesin-related protein, KIF1Bbeta2. KIF1Bbeta2 is a member of the KIF1 subfamily of kinesin-related proteins, and consists of an amino terminal KIF1B-type motor domain followed by a tail region highly similar to that of KIF1A. CHP binds to regions adjacent to the motor domains of KIF1Bbeta2 and KIF1B, but not to those of the other KIF1 family members, KIF1A and KIF1C. Immunostaining of neuronal cells showed that a significant portion of KIF1Bbeta2 is co-localized with synaptophysin, a marker protein for synaptic vesicles, but not with a mitochondria-staining dye. Subcellular fractionation analysis indicated the co-localization of KIF1Bbeta2 with synaptophysin. These results suggest that KIF1Bbeta2, a novel CHP-interacting molecular motor, mediates the transport of synaptic vesicles in neuronal cells.     

Kinesin superfamily proteins and their various functions and dynamics

Kinesin superfamily proteins (KIFs) are motor proteins that transport membranous organelles and macromolecules fundamental for cellular functions along microtubules. Their roles in transport in axons and dendrites have been studied extensively, but KIFs are also used in intracellular transport in general. Recent findings have revealed that in many cases, the specific interaction of cargoes and motors is mediated via adaptor/scaffolding proteins. Cargoes are sorted to precise destinations, such as axons or dendrites. KIFs also participate in polarized transport in epithelial cells as shown in the apical transport of annexin XIIIb-containing vesicles by KIFC3. KIFs play important roles in higher order neuronal activity; transgenic mice overexpressing KIF17, which transports N-methyl-d-asp (NMDA) receptors to dendrites, show enhanced memory and learning. KIFs also play significant roles in neuronal development and brain wiring: KIF2A suppresses elongation of axon collaterals by its unique microtubule-depolymerizing activity. X-ray crystallography has revealed the structural uniqueness of KIF2 underlying the microtubule-depolymerizing activity. In addition, single molecule biophysics and optical trapping have shown that the motility of monomeric KIF1A is caused by biased Brownian movement, and X-ray crystallography has shown how the conformational changes occur for KIF1A to move during ATP hydrolysis. These multiple approaches in analyzing KIF functions will illuminate many basic mechanisms underlying intracellular events and will be a very promising and fruitful area for future studies.     

A novel mouse kinesin of the UNC-104/KIF1 subfamily encoded by the Kif1b gene

Kinesin and kinesin-related proteins are microtubule-dependent motor proteins that transport organelles. We have cloned and sequenced a full-length 9924 bp mouse cDNA for a new kinesin of the UNC-104/KIF1 subfamily. Northern blot analysis of mouse RNAs detected high levels of a 10 kb mRNA in brain and eye, but lower levels in other tissues. Human RNA dot-blot analysis detected this mRNA in all tissues examined, although at different levels. The overall structure of the new kinesin (predicted size 204 kDa) was most similar to mouse KIF1A; however, 2.1 kb of the 5' portion of the cDNA were identical to the published sequence for KIF1B (Nangaku, M., Sato-Yoshitake, R., Okada, Y., Noda, Y., Takemura, R., Yamazaki, H., Hirokawa, N., 1994. KIF1B, a novel microtubule plus end-directed monomeric motor protein for transport of mitochondria. Cell 79, 1209-1220). We localized the Kif1b gene to the distal end of mouse Chromosome 4 by haplotype analysis of an interspecific backcross from The Jackson Laboratory. We had previously mapped the gene for the novel kinesin to the same location (Gong, T.-W.L., Burmeister, M., Lomax, M.I., 1996b. The novel gene D4Mille maps to mouse Chromosome 4 and human Chromosome 1p36. Mamm. Genome 7, 790-791). We conclude, therefore, that the Kif1b gene generates two major kinesin isoforms by alternative splicing. The shorter 7.8 kb mRNA encodes a 130 kDa kinesin, KIF1Bp130, whereas the 10 kb mRNA encodes a 204 kDa kinesin, KIF1Bp204. In addition, alternative splicing of two exons in the conserved region adjacent to the motor domain generates four different isoforms of each kinesin, leading to eight kinesin isoforms derived from the Kif1b gene.     

Charcot-Marie-Tooth disease type 2A caused by mutation in a microtubule motor KIF1Bbeta

The kinesin superfamily motor protein KIF1B has been shown to transport mitochondria. Here, we describe an isoform of KIF1B, KIF1Bbeta, that is distinct from KIF1B in its cargo binding domain. KIF1B knockout mice die at birth from apnea due to nervous system defects. Death of knockout neurons in culture can be rescued by expression of the beta isoform. The KIF1B heterozygotes have a defect in transporting synaptic vesicle precursors and suffer from progressive muscle weakness similar to human neuropathies. Charcot-Marie-Tooth disease type 2A was previously mapped to an interval containing KIF1B. We show that CMT2A patients contain a loss-of-function mutation in the motor domain of the KIF1B gene. This is clear indication that defects in axonal transport due to a mutated motor protein can underlie human peripheral neuropathy.     

Specific KIF1A–adaptor interactions control selective cargo recognition

Intracellular transport in neurons is driven by molecular motors that carry many different cargos along cytoskeletal tracks in axons and dendrites. Identifying how motors interact with specific types of transport vesicles has been challenging. Here, we use engineered motors and cargo adaptors to systematically investigate the selectivity and regulation of kinesin-3 family member KIF1A–driven transport of dense core vesicles (DCVs), lysosomes, and synaptic vesicles (SVs). We dissect the role of KIF1A domains in motor activity and show that CC1 regulates autoinhibition, CC2 regulates motor dimerization, and CC3 and PH mediate cargo binding. Furthermore, we identify that phosphorylation of KIF1A is critical for binding to vesicles. Cargo specificity is achieved by specific KIF1A adaptors; MADD/Rab3GEP links KIF1A to SVs, and Arf-like GTPase Arl8A mediates interactions with DCVs and lysosomes. We propose a model where motor dimerization, posttranslational modifications, and specific adaptors regulate selective KIF1A cargo trafficking.     

KIF1A mediates axonal transport of BACE1 and identification of independently moving cargoes in living SCG neurons

Neurons rely heavily on axonal transport to deliver materials from the sites of synthesis to the axon terminals over distances that can be many centimetres long. KIF1A is the neuron‐specific kinesin with the fastest reported anterograde motor activity. Previous studies have shown that KIF1A transports a subset of synaptic proteins, neurofilaments and dense‐core vesicles. Using two‐colour live imaging, we showed that beta‐secretase 1 (BACE1)‐mCherry moves together with KIF1A‐GFP in both the anterograde and retrograde directions in superior cervical ganglions (SCG) neurons. We confirmed that KIF1A is functionally required for BACE1 transport by using KIF1A siRNA and a KIF1A mutant construct (KIF1A‐T312M) to impair its motor activity. We further identified several cargoes that have little or no co‐migration with KIF1A‐GFP and also move independently from BACE1‐mCherry. Together, these findings support a primary role for KIF1A in the anterograde transport of BACE1 and suggest that axonally transported cargoes are sorted into different classes of carrier vesicles in the cell body and are transported by cargo‐specific motor proteins through the axon.      

High-resolution cryo-EM maps show the nucleotide binding pocket of KIF1A in open and closed conformations

Kinesin is an ATP-driven microtubule (MT)-based motor fundamental to organelle transport. Although a number of kinesin crystal structures have been solved, the structural evidence for coupling between the bound nucleotide and the conformation of kinesin is elusive. In addition, the structural basis of the MT-induced ATPase activity of kinesin is not clear because of the absence of the MT in the structure. Here, we report cryo-electron microscopy structures of the monomeric kinesin KIF1A-MT complex in two nucleotide states at about 10 A resolution, sufficient to reveal the secondary structure. These high-resolution maps visualized clear structural changes that suggest a mechanical pathway from the nucleotide to the neck linker via the motor core rotation. In addition, new nucleotide binding pocket conformations are observed that are different from X-ray crystallographic structures; it is closed in the 5'-adenylyl-imidodiphosphate state, but open in the ADP state. These results suggest a structural model of biased diffusion movement of monomeric kinesin motor.     

Stalk region of kinesin-related protein Unc104 has moderate ability to form coiled-coil dimer

Unc104/KIF1A, a kinesin family member, is reported to be monomeric in solution, though its polypeptide has regions that potentially form coiled coils. For a better understanding of the mechanism underlying Unc104/KIF1A's motility, it is important to evaluate the dimerization ability of this protein. The CD measurement of relevant segments of Caenorhabditis elegans Unc104 indicated that peptides having a common region (N358-K379) showed spectra characteristic to an alpha-helix. Dimerization by coiled-coil formation was confirmed by analytical ultracentrifugation. By analyzing the concentration dependence of the CD spectra, the monomer-dimer dissociation constant, Kd, of (N354-E388) was estimated to be about 5 microM, which is considerably larger than that of the corresponding segment of human kinesin (62 nM). Though its dimerization ability is rather moderate, Unc104/KIF1A could nonetheless dimerize and therefore could move by the same mechanism as human kinesin when the concentration of Unc104 is high due to, e.g., local crowding. This suggests that the motility could be controlled by the concentration of the motor protein.     

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

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. The results indicate that microtubule collision at 11.4 μm⁻¹ s⁻¹ coupled with ADP release at 78 s⁻¹ are fast steps for homodimeric KIF3AA. In contrast, KIF3CC exhibits much slower microtubule association at 2.1 μm⁻¹ s⁻¹ and ADP release at 8 s⁻¹. For KIF3AC, microtubule association at 6.6 μm⁻¹ s⁻¹ and ADP release at 51 s⁻¹ are intermediate between the constants for KIF3AA and KIF3CC. These results indicate that either KIF3A or KIF3C can initiate the processive run. Surprisingly, the kinetics of the initial event of microtubule collision followed by ADP release for KIF3AC is not equivalent to 1:1 mixtures of KIF3AA plus KIF3CC homodimers at the same motor concentration. These results reveal that the intermolecular communication within the KIF3AC heterodimer modulates entry into the processive run regardless of whether the run is initiated by the KIF3A or KIF3C motor domain.     

Association of the kinesin motor KIF1A with the multimodular protein liprin-alpha

Liprin-alpha/SYD-2 is a multimodular scaffolding protein important for presynaptic differentiation and postsynaptic targeting of alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid glutamate receptors. However, the molecular mechanisms underlying these functions remain largely unknown. Here we report that liprin-alpha interacts with the neuron-specific kinesin motor KIF1A. KIF1A colocalizes with liprin-alpha in various subcellular regions of neurons. KIF1A coaccumulates with liprin-alpha in ligated sciatic nerves. KIF1A cofractionates and coimmunopreciptates with liprin-alpha and various liprin-alpha-associated membrane, signaling, and scaffolding proteins including alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptors, GRIP/ABP, RIM, GIT1, and beta PIX. These results suggest that liprin-alpha functions as a KIF1A receptor, linking KIF1A to various liprin-alpha-associated proteins for their transport in neurons.     

Characterization of the movement of the kinesin motor KIF1A in living cultured neurons

KIF1A is a kinesin motor known to transport synaptic vesicle precursors in neuronal axons, but little is known about whether KIF1A mediates fast and processive axonal transport in vivo. By monitoring movements of EGFP-labeled KIF1A in living cultured hippocampal neurons, we determined the characteristics of KIF1A movements. KIF1A particles moved anterogradely along the neurites with an average velocity of 1.0 microm/s. The movements of KIF1A were highly processive, with an average duration of persistent anterograde movement of 11 s. Some KIF1A particles (17%) exhibited retrograde movements of 0.72 microm/s, although overall particle movement was in the anterograde direction. The anterograde movement of KIF1A, however, did not lead to a detectable accumulation of KIF1A in the periphery of neurons, suggesting that there are mechanisms inhibiting the peripheral accumulation of KIF1A. These results suggest that KIF1A mediates neuronal transport at a high velocity and processivity in vivo.     

Novel dendritic kinesin sorting identified by different process targeting of two related kinesins: KIF21A and KIF21B

Neurons use kinesin and dynein microtubule-dependent motor proteins to transport essential cellular components along axonal and dendritic microtubules. In a search for new kinesin-like proteins, we identified two neuronally enriched mouse kinesins that provide insight into a unique intracellular kinesin targeting mechanism in neurons. KIF21A and KIF21B share colinear amino acid similarity to each other, but not to any previously identified kinesins outside of the motor domain. Each protein also contains a domain of seven WD-40 repeats, which may be involved in binding to cargoes. Despite the amino acid sequence similarity between KIF21A and KIF21B, these proteins localize differently to dendrites and axons. KIF21A protein is localized throughout neurons, while KIF21B protein is highly enriched in dendrites. The plus end-directed motor activity of KIF21B and its enrichment in dendrites indicate that models suggesting that minus end-directed motor activity is sufficient for dendrite specific motor localization are inadequate. We suggest that a novel kinesin sorting mechanism is used by neurons to localize KIF21B protein to dendrites since its mRNA is restricted to the cell body.     

Delineation of the TRAK binding regions of the kinesin-1 motor proteins

Understanding specific cargo distribution in differentiated cells is a major challenge. Trafficking kinesin proteins (TRAKs) are kinesin adaptors. They bind the cargo binding domain of kinesin-1 motor proteins forming a link between the motor and their cargoes. To refine the TRAK1/2 binding sites within the kinesin-1 cargo domain, rationally designed C-terminal truncations of KIF5A and KIF5C were generated and their co-association with TRAK1/2 determined by quantitative co-immunoprecipitations following co-expression in mammalian cells. Three contributory regions forming the TRAK2 binding site within KIF5A and KIF5C cargo binding domains were delineated. Differences were found between TRAK1/2 with respect to association with KIF5A.