Yeast Kar3 is a minus-end microtubule motor protein that destabilizes microtubules 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 requirement for the protein both during mating and in mitosis. DNA sequence analysis predicts that Kar3 is a microtubule motor protein related to kinesin, but with the motor domain at the C-terminus of the protein rather than the N-terminus as in kinesin heavy chain. We have expressed Kar3 as a fusion protein with glutathione S-transferase (GST) and determined the in vitro motility properties of the bacterially expressed protein. The GST-Kar3 fusion protein bound to a coverslip translocates microtubules in gliding assays with a velocity of 1-2 microns/min and moves towards microtubule minus ends, unlike kinesin but like kinesin-related Drosophila ncd. Taxol-stabilized microtubules bound to GST-Kar3 on a coverslip shorten as they glide, resulting in faster lagging end, than leading end, velocities. Comparison of lagging and leading end velocities with velocities of asymmetrical axoneme-microtubule complexes indicates that microtubules shorten preferentially from the lagging or minus ends. The minus end-directed translocation and microtubule bundling of GST-Kar3 is consistent with models in which the Kar3 protein crosslinks internuclear microtubules and mediates nuclear fusion by moving towards microtubule minus ends, pulling the two nuclei together. In mitotic cells, the minus end motility of Kar3 could move chromosomes polewards, either by attaching to kinetochores and moving them polewards along microtubules, or by attaching to kinetochore microtubules and pulling them polewards along other polar microtubules.(ABSTRACT TRUNCATED AT 250 WORDS)
Endow
S A
SA
Department of Microbiology, Duke University Medical Center, Durham, NC 27710.
Kang
S J
SJ
Satterwhite
L L
LL
Rose
M D
MD
Skeen
V P
VP
Salmon
E D
ED
D000255:Adenosine Triphosphate / Q000494:pharmacology; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000494:pharmacology; D008857:Microscopy, Interference / Q000379:methods; D008868:Microtubule Proteins / Q000235:genetics / Q000378:metabolism*; D008869:Microtubule-Associated Proteins; D008870:Microtubules / Q000187:drug effects / Q000502:physiology*; D008954:Models, Biological; D017239:Paclitaxel; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins
D005656:Fungal Proteins; C063504:KAR3 protein, S cerevisiae; D008868:Microtubule Proteins; D008869:Microtubule-Associated Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C036852:magnesium GTP; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D017239:Paclitaxel