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 res...

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Main Authors:	Shirasu-Hiza, Mimi, Coughlin, Peg, Mitchison, Tim
Format:	Online
Language:	English
Published:	The Rockefeller University Press 2003
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2172913/

id	pubmed-2172913
recordtype	oai_dc
spelling	pubmed-21729132008-05-01 Identification of XMAP215 as a microtubule-destabilizing factor in Xenopus egg extract by biochemical purification Shirasu-Hiza, Mimi Coughlin, Peg Mitchison, Tim Article 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. The Rockefeller University Press 2003-04-28 /pmc/articles/PMC2172913/ /pubmed/12719474 http://dx.doi.org/10.1083/jcb.200211095 Text en Copyright © 2003, The Rockefeller University Press This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Shirasu-Hiza, Mimi Coughlin, Peg Mitchison, Tim
spellingShingle	Shirasu-Hiza, Mimi Coughlin, Peg Mitchison, Tim Identification of XMAP215 as a microtubule-destabilizing factor in Xenopus egg extract by biochemical purification
author_facet	Shirasu-Hiza, Mimi Coughlin, Peg Mitchison, Tim
author_sort	Shirasu-Hiza, Mimi
title	Identification of XMAP215 as a microtubule-destabilizing factor in Xenopus egg extract by biochemical purification
title_short	Identification of XMAP215 as a microtubule-destabilizing factor in Xenopus egg extract by biochemical purification
title_full	Identification of XMAP215 as a microtubule-destabilizing factor in Xenopus egg extract by biochemical purification
title_fullStr	Identification of XMAP215 as a microtubule-destabilizing factor in Xenopus egg extract by biochemical purification
title_full_unstemmed	Identification of XMAP215 as a microtubule-destabilizing factor in Xenopus egg extract by biochemical purification
title_sort	identification of xmap215 as a microtubule-destabilizing factor in xenopus egg extract by biochemical purification
description	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.
publisher	The Rockefeller University Press
publishDate	2003
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2172913/
_version_	1611425098448764928

Identification of XMAP215 as a microtubule-destabilizing factor in Xenopus egg extract by biochemical purification

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