The Caenorhabditis elegans Kinesin-3 Motor UNC-104/KIF1A Is Degraded upon Loss of Specific Binding to Cargo

National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore, India.
PLoS Genetics (Impact Factor: 7.53). 11/2010; 6(11):e1001200. DOI: 10.1371/journal.pgen.1001200
Source: PubMed

ABSTRACT UNC-104/KIF1A is a Kinesin-3 motor that transports synaptic vesicles from the cell body towards the synapse by binding to PI(4,5)P(2) through its PH domain. The fate of the motor upon reaching the synapse is not known. We found that wild-type UNC-104 is degraded at synaptic regions through the ubiquitin pathway and is not retrogradely transported back to the cell body. As a possible means to regulate the motor, we tested the effect of cargo binding on UNC-104 levels. The unc-104(e1265) allele carries a point mutation (D1497N) in the PI(4,5)P(2) binding pocket of the PH domain, resulting in greatly reduced preferential binding to PI(4,5)P(2)in vitro and presence of very few motors on pre-synaptic vesicles in vivo. unc-104(e1265) animals have poor locomotion irrespective of in vivo PI(4,5)P(2) levels due to reduced anterograde transport. Moreover, they show highly reduced levels of UNC-104 in vivo. To confirm that loss of cargo binding specificity reduces motor levels, we isolated two intragenic suppressors with compensatory mutations within the PH domain. These show partial restoration of in vitro preferential PI(4,5)P(2) binding and presence of more motors on pre-synaptic vesicles in vivo. These animals show improved locomotion dependent on in vivo PI(4,5)P(2) levels, increased anterograde transport, and partial restoration of UNC-104 protein levels in vivo. For further proof, we mutated a conserved residue in one suppressor background. The PH domain in this triple mutant lacked in vitro PI(4,5)P(2) binding specificity, and the animals again showed locomotory defects and reduced motor levels. All allelic variants show increased UNC-104 levels upon blocking the ubiquitin pathway. These data show that inability to bind cargo can target motors for degradation. In view of the observed degradation of the motor in synaptic regions, this further suggests that UNC-104 may get degraded at synapses upon release of cargo.

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Available from: Raghu Metpally, Sep 25, 2015
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    • "(not significant) are comparisons of each strain to wild type. a decrease in anterograde velocity (Kumar et al. 2010; Maeder et al. 2014). Since the ce782 mutation is in the motor domain, which is on the opposite end of the protein from the cargo binding domain, it is possible that the ce782 mutation also indirectly affects dynein motor activity in these bidirectionally moving vesicles. "
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    ABSTRACT: The functional integrity of neurons requires the bidirectional active transport of synaptic vesicles (SVs) in axons. The kinesin motor KIF1A transports SVs from somas to stable SV clusters at synapses, while dynein moves them in the opposite direction. However, it is unclear how SV transport is regulated and how SVs at clusters interact with motor proteins. We addressed these questions by isolating a rare temperature-sensitive allele of Caenorhabditis elegans unc-104 (KIF1A) that allowed us to manipulate SV levels in axons and dendrites. Growth at 20° and 14° resulted in locomotion rates that were 3 and 50% of wild type, respectively, with similar effects on axonal SV levels. Corresponding with the loss of SVs from axons, mutants grown at 14° and 20° showed a 10-and 24-fold dynein-dependent accumulation of SVs in their dendrites. Mutants grown at 14° and switched to 25° showed an abrupt irreversible 50% decrease in locomotion and a 50% loss of SVs from the synaptic region 12-hr post-shift, with no further decreases at later time points, suggesting that the remaining clustered SVs are stable and resistant to retrograde removal by dynein. The data further showed that the synapse-assembly proteins SYD-1, SYD-2, and SAD-1 protected SV clusters from degradation by motor proteins. In syd-1, syd-2, and sad-1 mutants, SVs accumulate in an UNC-104-dependent manner in the distal axon region that normally lacks SVs. In addition to their roles in SV cluster stability, all three proteins also regulate SV transport.
    Genetics 09/2015; 201(1):91-116. DOI:10.1534/genetics.115.177337 · 5.96 Impact Factor
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    • "Please cite this article in press as: Neumann and Hilliard, Loss of MEC-17 Leads to Microtubule Instability and Axonal Degeneration, Cell Reports (2014), tagged version of UNC-104/kinesin-3 (Kumar et al., 2010), one of the main motor molecules responsible for transport of synaptic vesicles to presynaptic loci (Hall and Hedgecock, 1991). Wildtype animals exhibited a consistent distribution of fluorescence in PLM, with a smooth increase in expression along the axon in a proximal-to-distal fashion, and pooling at the distal end and at presynaptic sites (Figure 3A). "
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    ABSTRACT: Axonal degeneration arises as a consequence of neuronal injury and is a common hallmark of a number of neurodegenerative diseases. However, the genetic causes and the cellular mechanisms that trigger this process are still largely unknown. Based on forward genetic screening in C. elegans, we have identified the α-tubulin acetyltransferase gene mec-17 as causing spontaneous, adult-onset, and progressive axonal degeneration. Loss of MEC-17 leads to microtubule instability, a reduction in mitochondrial number, and disrupted axonal transport, with altered distribution of both mitochondria and synaptic components. Furthermore, mec-17-mediated axonal degeneration occurs independently from its acetyltransferase domain; is enhanced by mutation of coel-1, a tubulin-associated molecule; and correlates with the animal's body length. This study therefore identifies a critical role for the conserved microtubule-associated protein MEC-17 in preserving axon integrity and preventing axonal degeneration.
    Cell Reports 12/2013; 6(1). DOI:10.1016/j.celrep.2013.12.004 · 8.36 Impact Factor
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    • "Nerve ligature experiments indicate that kinesin-3 motors are not recycled by cytoplasmic dynein-dependent transport as they do not accumulate on the distal side of the ligature [18]. In addition, a recent study in C. elegans suggested that the kinesin-3 motor UNC-104 is not retrogradely transported by cytoplasmic dynein [97]. However, these results appear to contradict previous work from the same group showing that mutations in various cytoplasmic dynein subunits caused an accumulation of UNC-104 at the ends of neuronal processes [98]. "
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    ABSTRACT: Kinesin motors drive the long-distance anterograde transport of cellular components along microtubule tracks. Kinesin-dependent transport plays a critical role in neurogenesis and neuronal function due to the large distance separating the soma and nerve terminal. The fate of kinesin motors after delivery of their cargoes is unknown but has been postulated to involve degradation at the nerve terminal, recycling via retrograde motors, and/or recycling via diffusion. We set out to test these models concerning the fate of kinesin-1 motors after completion of transport in neuronal cells. We find that kinesin-1 motors are neither degraded nor returned by retrograde motors. By combining mathematical modeling and experimental analysis, we propose a model in which the distribution and recycling of kinesin-1 motors fits a "loose bucket brigade" where individual motors alter between periods of active transport and free diffusion within neuronal processes. These results suggest that individual kinesin-1 motors are utilized for multiple rounds of transport.
    PLoS ONE 09/2013; 8(9):e76081. DOI:10.1371/journal.pone.0076081 · 3.23 Impact Factor
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