Department of Physics, Oregon State University, Corvallis, Oregon.
Department of Physics, Oregon State University, Corvallis, Oregon; School of Physics and Electronics, Henan University, Kaifeng, Henan, China.
Biophys J. 2019 Apr 2;116(7):1270-1281. doi: 10.1016/j.bpj.2019.02.019. Epub 2019 Feb 28.
Phragmoplast-associated kinesin-related protein 2 (PAKRP2) is an orphan kinesin in Arabidopsis thaliana that is thought to transport vesicles along phragmoplast microtubules for cell plate formation. Here, using single-molecule fluorescence microscopy, we show that PAKRP2 is the first orphan kinesin to exhibit processive plus-end-directed motility on single microtubules as individual homodimers. Our results show that PAKRP2 processivity is achieved despite having an exceptionally long (32 residues) neck linker. Furthermore, using high-resolution nanoparticle tracking, we find that PAKRP2 steps via a hand-over-hand mechanism that includes frequent side steps, a prolonged diffusional search of the tethered head, and tight coupling of the ATP hydrolysis cycle to the forward-stepping cycle. Interestingly, truncating the PAKRP2 neck linker to 14 residues decreases the run length of PAKRP2; thus, the long neck linker enhances the processive behavior. Based on the canonical model of kinesin stepping, such a long neck linker is expected to decrease the processivity and disrupt the coupling of ATP hydrolysis to forward stepping. Therefore, we conclude that PAKRP2 employs a noncanonical strategy for processive motility, wherein a long neck linker is coupled with a slow ATP hydrolysis rate to allow for an extended diffusional search during each step without sacrificing processivity or efficiency.
Phragmoplast 相关的驱动蛋白相关蛋白 2（PAKRP2）是拟南芥中的一种孤儿驱动蛋白，被认为是用于细胞板形成的沿着成膜体微管运输囊泡的蛋白。在这里，我们使用单分子荧光显微镜，首次证明 PAKRP2 作为单个同源二聚体在单个微管上表现出连续的正极定向运动，这是第一个表现出这种性质的孤儿驱动蛋白。我们的结果表明，尽管 PAKRP2 的颈环连接体异常长（32 个残基），但其连续性是可以实现的。此外，我们使用高分辨率纳米颗粒跟踪技术发现，PAKRP2 通过一种手对手的机制进行步移，其中包括频繁的侧步、被束缚头部的延长扩散搜索以及 ATP 水解循环与前向步移循环的紧密偶联。有趣的是，将 PAKRP2 的颈环连接体截断至 14 个残基会缩短 PAKRP2 的运行长度；因此，长的颈环连接体增强了连续性行为。根据驱动蛋白步移的典型模型，这种长的颈环连接体预计会降低连续性并破坏 ATP 水解与向前步移的偶联。因此，我们得出结论，PAKRP2 采用了一种非典型的连续性运动策略，其中长的颈环连接体与缓慢的 ATP 水解速率相耦合，允许在每个步移期间进行扩展的扩散搜索，而不会牺牲连续性或效率。
