Abstract
Motor proteins of the conserved kinesin-14 family have important roles in mitotic spindle organization and chromosome segregation. Previous studies have indicated that kinesin-14 motors are non-processive enzymes, working in the context of multi-motor ensembles that collectively organize microtubule networks. In this study, we show that the yeast kinesin-14 Kar3 generates processive movement as a heterodimer with the non-motor proteins Cik1 or Vik1. By analyzing the single-molecule properties of engineered motors, we demonstrate that the non-catalytic domain has a key role in the motility mechanism by acting as a 'foothold' that allows Kar3 to bias translocation towards the minus end. This mechanism rivals the speed and run length of conventional motors, can support transport of the Ndc80 complex in vitro and is critical for Kar3 function in vivo. Our findings provide an example for a non-conventional translocation mechanism and can explain how Kar3 substitutes for key functions of Dynein in the yeast nucleus.
Highlights
Motors of the kinesin family are ubiquitous enzymes essential for intracellular transport along microtubules in eukaryotes
To study Kar3 motors at the single molecule level, we developed a protocol to express and purify full-length kinesin-14 heterodimers from Saccharomyces cerevisiae using affinity tagged Cik1 and Kar3
We further characterized the oligomeric state of full-length Cik1–Kar3 motors by performing low-angle Pt/C rotary shadowing electron microscopy on peak fractions from the gel filtration experiments
Summary
Motors of the kinesin family are ubiquitous enzymes essential for intracellular transport along microtubules in eukaryotes. In analogy to other enzymes, the term ‘processivity’ describes the ability of individual motor molecules to undergo multiple catalytic cycles—and translocate—before releasing from the microtubule. Kinesin-14 family members, exemplified by the Drosophila Ncd motor, are common examples for nonprocessive kinesins (Case et al, 1997; Foster and Gilbert, 2000). They generate motility through the minus-end-directed rotational movement of a coiled-coil mechanical element that occurs upon ATP binding (Endres et al, 2006).
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