RT Journal Article SR Electronic T1 Kinesin-2 from C. reinhardtii is an atypically fast and auto-inhibited motor that is activated by heterotrimerization for intraflagellar transport JF bioRxiv FD Cold Spring Harbor Laboratory SP 855940 DO 10.1101/855940 A1 Punam Sonar A1 Wiphu Youyen A1 Augustine Cleetus A1 Pattipong Wisanpitayakorn A1 Iman S. Mousavi A1 Willi L. Stepp A1 William O. Hancock A1 Erkan Tüzel A1 Zeynep Ökten YR 2019 UL http://biorxiv.org/content/early/2019/11/26/855940.abstract AB The construction and function of virtually all cilia require the universally conserved process of Intraflagellar Transport (IFT) [1, 2]. During the atypically fast IFT in the green alga C. reinhardtii, up to ten kinesin-2 motors ‘line up’ in a tight assembly on the trains [3], provoking the question of how these motors coordinate their action to ensure smooth and fast transport along the flagellum without standing in each other’s way. Here, we show that the heterodimeric FLA8/10 kinesin-2 alone is responsible for the atypically fast IFT in C. reinhardtii. Notably, in single-molecule studies, FLA8/10 moved at speeds matching those of in vivo IFT [4], but additionally displayed a slow velocity distribution, indicative of auto-inhibition. Addition of the KAP subunit to generate the heterotrimeric FLA8/10/KAP relieved this inhibition, thus providing a mechanistic rationale for heterotrimerization with the KAP subunit in fully activating FLA8/10 for IFT in vivo. Finally, we link fast FLA8/10 and slow KLP11/20 kinesin-2 from C. reinhardtii and C. elegans through a DNA tether to understand the molecular underpinnings of motor coordination during IFT in vivo. For motor pairs from both species, the co-transport velocities very nearly matched the single-molecule velocities, and the complexes both spent roughly 80% of the time with only one of the two motors attached to the microtubule. Thus, irrespective of phylogeny and kinetic properties, kinesin-2 motors prefer to work alone without sacrificing efficiency. Our findings thus offer a simple mechanism for how efficient IFT is achieved across diverse organisms despite being carried out by motors with different properties.