Analysis of the in vitro activity and cargo binding characteristics of kinesin-2.

摘要

Kinesin-2 is an abundant motor that functions in a variety of cellular pathways. It is a heterotrimeric protein consisting of two motor subunit combinations (A/B and A/C) bound to a non-motor subunit (KAP3). Many of the details of how kinesin-2 activity and cargo selection is regulated remain poorly understood. An interaction between KAP3 of kinesin-2 and dynactin, the dynein activating and cargo linking complex, has been described. The goals of my thesis were to optimize a method of purification of native, vertebrate kinesin-2 for biochemical and enzymatic analyses and to prepare a series of KAP3 fragments that could be used in a detailed assessment of the kinesin-2: dynactin interaction.; Modification of the anion exchange chromatography conditions for chick embryo brain kinesin-2 was effective at separating the closely related A/B and A/C motor isoforms from each other. This allowed for an in depth analysis of these two forms of kinesin-2. The ability to purify intact, native kinesin-2 gave me the raw materials to investigate motor activity and the effects of dynactin at the single-molecule level. This line of research led me to be the first to quantify kinesin-2 processivity and compare the effects of dynactin on its processivity to what is seen with other motors. Finally, I defined an interaction domain of KAP3 on p150Glued, the dynein binding subunit of dynactin, and identified other dynactin subunits that may contribute to kinesin-2 binding. My results suggest that single dynactin molecules may be able to bind both kinesin-2 and dynein simultaneously. I also discovered that kinesin-2 can bind actin, which may point to actin as being a novel cargo/cargo linking system for kinesin-2. This result is in line with kinesin-2's known cellular functions in cilliary/flagellar maintenance and neurite extension.; Overall, I have worked to help elucidate the biochemical, enzymatic and cargo binding properties of kinesin-2. My work serves as a basis for understanding the complexities of this important kinesin family motor and hopefully will act to inspire future research into its importance at the molecular, cellular and organismal levels.