The overarching goal is to gain critical insights into the fundamentals of motor structure and function and to extrapolate this understanding to the inner workings of the cell. Kinesin superfamily members share a common catalytic domain yet participate in a wide range of cellular functions including intracellular transport, mitosis and meiosis, regulation of microtubule dynamics for remodeling of the cytoskeleton, and generation of cell polarity. Sequence differences modify the mechanochemistry and microtubule interactions that are critical for the specific function. The goal of this proposal is to establishthe mechanistic and structural features shared by Kinesin-14 Kar3Cik1, Kar3Vik1, and Ncd and at the same time to reveal unique features that result in functional specificity. Members of the Kinesin-14 subfamily are the only kinesins known to promote microtubule minus-end-directed force generation, and these motors are not processive. In contrast, members of Kinesin-1, 2, 5, 7 subfamilies generate microtubule plus-end-directed force, and these molecular motors are processive. Conventional Kinesin-1, Kinesin-5 Eg5, and Kinesin-7 CENP-E generate motors from the same gene product, yet the functional catalytic dimer for Kinesin-2 arises from two different gene products. Therefore, what is the selective advantage of heterodimeric catalytic enzymes for in vivo function and how is head-head communication established to modulate interactions with the microtubule lattice and/or microtubule end? The research proposed evaluates heterodimeric Kar3Cik1 and Kar3Vik1 in comparison to homodimeric Ncd, and heterodimeric Kinesin-2 KIFAB and KIFAC in comparison to other processive homodimeric kinesins including conventional Kinesin-1, Eg5, and CENP-E. Experimental approaches include pre-steady state kinetic methodologies, fluorescence microscopy of microtubule-motor complexes, X-ray crystallography, and cryo- electron microscopy and tomography. This comprehensive analysis of Kinesin-14 and Kinesin-2 members will provide new insights to understand the mechanochemistry that underlies structure-function relationships required for cellular organization and function.