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PMID:21079789

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Citation

Kumar, J, Choudhary, BC, Metpally, R, Zheng, Q, Nonet, ML, Ramanathan, S, Klopfenstein, DR and Koushika, SP (2010) The Caenorhabditis elegans Kinesin-3 motor UNC-104/KIF1A is degraded upon loss of specific binding to cargo. PLoS Genet. 6:e1001200

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.

Links

PubMed PMC2973836 Online version:10.1371/journal.pgen.1001200

Keywords

Alleles; Animals; Animals, Genetically Modified; Caenorhabditis elegans/metabolism; Caenorhabditis elegans Proteins/chemistry; Caenorhabditis elegans Proteins/metabolism; Conserved Sequence/genetics; Genes, Suppressor; Green Fluorescent Proteins/metabolism; Kinesin/chemistry; Kinesin/metabolism; Mutant Proteins/chemistry; Mutant Proteins/metabolism; Nerve Tissue Proteins/chemistry; Nerve Tissue Proteins/metabolism; Neurons/cytology; Neurons/metabolism; Phenotype; Phosphatidylinositol 4,5-Diphosphate/metabolism; Protein Binding; Protein Processing, Post-Translational; Protein Structure, Tertiary; Protein Transport; Recombinant Fusion Proteins/metabolism; Synapses/metabolism; Synaptic Vesicles/metabolism; Ubiquitin/metabolism

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

CAEEL:UN104

enables

GO:0005546: phosphatidylinositol-4,5-bisphosphate binding

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

CAEEL:UN104

located_in

GO:0043025: neuronal cell body

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

CAEEL:UN104

enables

GO:0070273: phosphatidylinositol-4-phosphate binding

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

CAEEL:UN104

located_in

GO:0030424: axon

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

CAEEL:UN104

located_in

GO:0098793: presynapse

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete


See also

References

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