Localization of the kinesin-like protein Xklp2 to spindle poles requires a leucine zipper, a microtubule-associated protein, and dynein.
Xklp2 is a plus end-directed Xenopus kinesin-like protein localized at spindle poles and required for centrosome separation during spindle assembly in Xenopus egg extracts. A glutathione-S-transferase fusion protein containing the COOH-terminal domain of Xklp2 (GST-Xklp2-Tail) was previously found to localize to spindle poles (Boleti, H., E. Karsenti, and I. Vernos. 1996. Cell. 84:49-59). Now, we have examined the mechanism of localization of GST-Xklp2-Tail. Immunofluorescence and electron microscopy showed that Xklp2 and GST-Xklp2-Tail localize specifically to the minus ends of spindle pole and aster microtubules in mitotic, but not in interphase, Xenopus egg extracts. We found that dimerization and a COOH-terminal leucine zipper are required for this localization: a single point mutation in the leucine zipper prevented targeting. The mechanism of localization is complex and two additional factors in mitotic egg extracts are required for the targeting of GST-Xklp2-Tail to microtubule minus ends: (a) a novel 100-kD microtubule-associated protein that we named TPX2 (Targeting protein for Xklp2) that mediates the binding of GST-Xklp2-Tail to microtubules and (b) the dynein-dynactin complex that is required for the accumulation of GST-Xklp2-Tail at microtubule minus ends. We propose two molecular mechanisms that could account for the localization of Xklp2 to microtubule minus ends.
Wittmann
T
T
European Molecular Biology Laboratory, Cell Biology and Cell Biophysics Programs, D-69117 Heidelberg, Germany.
Boleti
H
H
Antony
C
C
Karsenti
E
E
Vernos
I
I
D000818:Animals; D001665:Binding Sites; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002457:Cell Extracts; D019281:Dimerization; D000072159:Dynactin Complex; D004398:Dyneins / Q000378:metabolism*; D006801:Humans; D016547:Kinesins / Q000378:metabolism*; D016350:Leucine Zippers; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D008941:Spindle Apparatus; D014981:Xenopus; D029867:Xenopus Proteins
D018797:Cell Cycle Proteins; D002457:Cell Extracts; D000072159:Dynactin Complex; C097723:KIF15 protein, Xenopus; D008869:Microtubule-Associated Proteins; D011993:Recombinant Fusion Proteins; D029867:Xenopus Proteins; D004398:Dyneins; D016547:Kinesins