Graduate Thesis Or Dissertation


Single-Molecule Characterization of the Stepping Mechanism of the Orphan Kinesin PAKRP2 Public Deposited

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  • The phragmoplast associated kinesin, PAKRP2, from the model organism Arabidopsis thaliana is categorized as an orphan, or ungrouped, kinesin due to structural divergences from the established 14 kinesin families. An early study on PAKRP2 found that it localized to the phragmoplast mid zone during the late stages of cell division and predicted it to be involved in vesicle transport of material to the developing cell wall. Many orphan kinesins have mutations in the microtubule and/or nucleotide binding sites in the motor domain that prevent them from being processive, which is a conserved feature of all transport kinesins. PAKRP2 contains a mutation in the nucleotide binding site but no investigation of its motility has ever been done. Using total internal reflection fluorescence microscopy and total internal reflection dark-field microscopy to perform single-molecule assays, we have characterized the motility of PAKRP2. We find that PAKRP2 is a highly processive motor that does not require clustering or aid from a nonmotor microtubule binding domain to achieve processivity, which makes it similar to other transport kinesins. Two separate stepping assays, with a gold nanoparticle attached to either the motor or tail domains, reveal that PAKRP2 steps in a noncanonical manner that includes small step sizes and frequent lateral steps. We predict the small step sizes are due to a transient intermediate step, wherein the tethered head diffusively searches for the next binding site while ATP hydrolysis occurs in the bound head. A coiled-coil prediction revealed that PAKRP2 contains an extraordinarily long neck linker (32 residues), which corroborates the idea of an extended 1 HB state in the PAKRP2 motility cycle. Additionally, the long neck linker does not adversely affect the interhead coupling, as is seen in other kinesins, and actually contributes to the processivity of the motor. Overall, this study demonstrates the first processive orphan kinesin and adds to the developing model of kinesin stepping.
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