Mitotic kinesin CENP-E promotes microtubule plus-end elongation.
Centromere protein CENP-E is a dimeric kinesin (Kinesin-7 family) with critical roles in mitosis, including establishment of microtubule (MT)-chromosome linkage and movement of mono-oriented chromosomes on kinetochore microtubules for proper alignment at metaphase [1-9]. We performed studies to test the hypothesis that CENP-E promotes MT elongation at the MT plus ends. A human CENP-E construct was engineered, expressed, and purified, and it yielded the CENP-E-6His dimeric motor protein. The results show that CENP-E promotes MT plus-end-directed MT gliding at 11 nm/s. The results from real-time microscopy assays indicate that 60.3% of polarity-marked MTs exhibited CENP-E-promoted MT plus-end elongation. The MT extension required ATP turnover, and MT plus-end elongation occurred at 1.48 μm/30 min. Immunolocalization studies revealed that 80.8% of plus-end-elongated MTs showed CENP-E at the MT plus end. The time dependence of CENP-E-promoted MT elongation in solution best fit a single exponential function (k(obs) = 5.1 s(-1)), which is indicative of a mechanism in which α,β-tubulin subunit addition is tightly coupled to ATP turnover. Based on these results, we propose that CENP-E, as part of its function in chromosome kinetochore-MT linkage, plays a direct role in MT elongation.
Sardar
Harjinder S
HS
Department of Biology, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
Luczak
Vincent G
VG
Lopez
Maria M
MM
Lister
Bradford C
BC
Gilbert
Susan P
SP
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D006801:Humans; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D002868:Chromosomal Proteins, Non-Histone; D011993:Recombinant Fusion Proteins; C068688:centromere protein E; D000255:Adenosine Triphosphate
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