YEAST KAR3 IS A MINUS-END MICROTUBULE MOTOR PROTEIN THAT DESTABILIZESMICROTUBULES PREFERENTIALLY AT THE MINUS ENDS

Mutants of the yeast Kar3 protein are defective in nuclear fusion, or karyogamy, during mating and show slow mitotic growth, indicating a re quirement for the protein both during mating and in mitosis. DNA seque nce analysis predicts that Kar3 is a microtubule motor protein related to kinesin, but with the motor domain at the C-terminus of the protei n rather than the N-terminus as in kinesin heavy chain. We have expres sed Kar3 as a fusion protein with glutathione S-transferase (GST) and determined the in vitro motility properties of the bacterially express ed protein. The GST-Kar3 fusion protein bound to a coverslip transloca tes microtubules in gliding assays with a velocity of 1-2 mu m/min and moves towards microtubule minus ends, unlike kinesin but like kinesin -related Drosophila ncd. Taxol-stabilized microtubules bound to GST-Ka r3 on a coverslip shorten as they glide, resulting in faster lagging e nd, than leading end, velocities. Comparison of lagging and leading en d velocities with velocities of asymmetrical axoneme-microtubule compl exes indicates that microtubules shorten preferentially from the laggi ng or minus ends. The minus end-directed translocation and microtubule bundling of GST-Kar3 is consistent with models in which the Kar3 prot ein crosslinks internuclear microtubules and mediates nuclear fusion b y moving towards microtubule minus ends, pulling the two nuclei togeth er. In mitotic cells, the minus end motility of Kar3 could move chromo somes polewards, either by attaching to kinetochores and moving them p olewards along microtubules, or by attaching to kinetochore microtubul es and pulling them polewards along other polar microtubules. The dest abilization at microtubule minus ends observed; in our in vitro studie s suggests that Kar3 may also function to promote microtubule depolyme rization at spindle pole bodies during karyogamy and mitosis. The minu s end polarity of Kar3 and ncd translocation, and the structural simil arity to Kar3 and ncd of kinesin-related proteins from Arabidopsis and mammalian cells, support the idea that a unique subfamily of the kine sin proteins consists of minus end-directed motors.