Identification of XMAP215 as a microtubule-destabilizing factor in Xenopus egg extract by biochemical purification.
Microtubules (MTs) polymerized with GMPCPP, a slowly hydrolyzable GTP analogue, are stable in buffer but are rapidly depolymerized in Xenopus egg extracts. This depolymerization is independent of three previously identified MT destabilizers (Op18, katanin, and XKCM1/KinI). We purified the factor responsible for this novel depolymerizing activity using biochemical fractionation and a visual activity assay and identified it as XMAP215, previously identified as a prominent MT growth-promoting protein in Xenopus extracts. Consistent with the purification results, we find that XMAP215 is necessary for GMPCPP-MT destabilization in extracts and that recombinant full-length XMAP215 as well as an NH2-terminal fragment have depolymerizing activity in vitro. Stimulation of depolymerization is specific for the MT plus end. These results provide evidence for a robust MT-destabilizing activity intrinsic to this microtubule-associated protein and suggest that destabilization may be part of its essential biochemical functions. We propose that the substrate in our assay, GMPCPP-stabilized MTs, serves as a model for the pause state of MT ends and that the multiple activities of XMAP215 are unified by a mechanism of antagonizing MT pauses.
Department of Cell Biology, Harvard Medical School, 250 Longwood Ave., Boston, MA 02115, USA. mshirasu@hms.harvard.edu
Mimi
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Shirasu-Hiza
Peg
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Coughlin
Tim
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Mitchison
D000818:Animals; D001681:Biological Assay; D002457:Cell Extracts / Q000737:chemistry* / Q000494:pharmacology*; D005260:Female; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D008868:Microtubule Proteins / Q000096:biosynthesis / Q000187:drug effects / Q000235:genetics; D008869:Microtubule-Associated Proteins / Q000302:isolation & purification* / Q000494:pharmacology; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism*; D008954:Models, Biological; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D029867:Xenopus Proteins; D014982:Xenopus laevis / Q000378:metabolism*
C486738:CKAP5 protein, Xenopus; D002457:Cell Extracts; D008868:Microtubule Proteins; D008869:Microtubule-Associated Proteins; D011993:Recombinant Fusion Proteins; D029867:Xenopus Proteins; C004805:5'-guanylylmethylenebisphosphonate; D006160:Guanosine Triphosphate