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J. Cell Biol.,
Volume 143, Number 3, November 2, 1998 673-685
* European Molecular Biology Laboratory, Cell Biology and Cell Biophysics Programs, D-69117 Heidelberg, Germany; 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.
Institut Pasteur, 75724 Paris Cedex 15, France; and § Institut Curie, 75248 Paris Cedex 05, France
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