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

Shirasu-Hiza M, Coughlin P, Mitchison T - J. Cell Biol. (2003)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Harvard Medical School, 250 Longwood Ave., Boston, MA 02115, USA. mshirasu@hms.harvard.edu

ABSTRACT
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.

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Proteins of 130 and 160 kD were enriched during the purification and consistently copeaked with activity. (A) Specific activity increased with each step of the purification, as did the prominence of p160 and p130 bands (arrows). Shown here is a silver-stained polyacrylamide gel containing fractions from the purification. Samples for the first seven lanes are listed as follows: mw, molecular weight markers; AS supe, 40% AS supernatant (5 μg protein; 4 U of specific activity); PS pool, phenyl sepharose pool of active fractions (4 μg; 10 U); hep pool, heparin pool (4.8 μg; 300 U); monoS 1, first monoS column (5 μg; 600 U); sup6, gel filtration/superose 6 (2.5 μg; 600 U). The remaining lanes (monoS2) represent fractions from the second monoS column; depolymerizing activity peaked in fractions 16, 17, and 18 (see B). Lanes loaded with these fractions each contain ∼1.3 μg of total protein and 1,260 U of specific activity. Arrows indicate p130 and p160 bands. Identity of the p130 bands (lower arrows) from these fractions was determined by mass spectrometry. (B) Activity profile for fractions for the monoS2 step. Relative activity was estimated for fractions 8–24 by serial titration in the depolymerization assay and is presented in this graph as arbitrary units per microgram protein. (C) p160 and p130 bands are NH2-terminal fragments of XMAP215. Western blots of high-speed supernatant (hss) and monoS2 fractions were probed for XMAP215 with NH2-terminal– or COOH-terminal–specific antibodies.
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fig2: Proteins of 130 and 160 kD were enriched during the purification and consistently copeaked with activity. (A) Specific activity increased with each step of the purification, as did the prominence of p160 and p130 bands (arrows). Shown here is a silver-stained polyacrylamide gel containing fractions from the purification. Samples for the first seven lanes are listed as follows: mw, molecular weight markers; AS supe, 40% AS supernatant (5 μg protein; 4 U of specific activity); PS pool, phenyl sepharose pool of active fractions (4 μg; 10 U); hep pool, heparin pool (4.8 μg; 300 U); monoS 1, first monoS column (5 μg; 600 U); sup6, gel filtration/superose 6 (2.5 μg; 600 U). The remaining lanes (monoS2) represent fractions from the second monoS column; depolymerizing activity peaked in fractions 16, 17, and 18 (see B). Lanes loaded with these fractions each contain ∼1.3 μg of total protein and 1,260 U of specific activity. Arrows indicate p130 and p160 bands. Identity of the p130 bands (lower arrows) from these fractions was determined by mass spectrometry. (B) Activity profile for fractions for the monoS2 step. Relative activity was estimated for fractions 8–24 by serial titration in the depolymerization assay and is presented in this graph as arbitrary units per microgram protein. (C) p160 and p130 bands are NH2-terminal fragments of XMAP215. Western blots of high-speed supernatant (hss) and monoS2 fractions were probed for XMAP215 with NH2-terminal– or COOH-terminal–specific antibodies.

Mentions: The CPP MT–depolymerizing factor was purified using seven steps: AS precipitation, phenyl sepharose, heparin, monoS, gel filtration, monoQ (pH 7.2), and a final monoS column. When fractions were separated by SDS-PAGE and silver stained, a set of polypeptides of ∼130 kD and a protein of ∼160 kD consistently coeluted with activity on the last two columns in the purification (Fig. 2, A and B , arrows). We estimated that specific activity was enriched several thousand fold by the final monoS step (Table I).


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

Shirasu-Hiza M, Coughlin P, Mitchison T - J. Cell Biol. (2003)

Proteins of 130 and 160 kD were enriched during the purification and consistently copeaked with activity. (A) Specific activity increased with each step of the purification, as did the prominence of p160 and p130 bands (arrows). Shown here is a silver-stained polyacrylamide gel containing fractions from the purification. Samples for the first seven lanes are listed as follows: mw, molecular weight markers; AS supe, 40% AS supernatant (5 μg protein; 4 U of specific activity); PS pool, phenyl sepharose pool of active fractions (4 μg; 10 U); hep pool, heparin pool (4.8 μg; 300 U); monoS 1, first monoS column (5 μg; 600 U); sup6, gel filtration/superose 6 (2.5 μg; 600 U). The remaining lanes (monoS2) represent fractions from the second monoS column; depolymerizing activity peaked in fractions 16, 17, and 18 (see B). Lanes loaded with these fractions each contain ∼1.3 μg of total protein and 1,260 U of specific activity. Arrows indicate p130 and p160 bands. Identity of the p130 bands (lower arrows) from these fractions was determined by mass spectrometry. (B) Activity profile for fractions for the monoS2 step. Relative activity was estimated for fractions 8–24 by serial titration in the depolymerization assay and is presented in this graph as arbitrary units per microgram protein. (C) p160 and p130 bands are NH2-terminal fragments of XMAP215. Western blots of high-speed supernatant (hss) and monoS2 fractions were probed for XMAP215 with NH2-terminal– or COOH-terminal–specific antibodies.
© Copyright Policy
Related In: Results  -  Collection

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fig2: Proteins of 130 and 160 kD were enriched during the purification and consistently copeaked with activity. (A) Specific activity increased with each step of the purification, as did the prominence of p160 and p130 bands (arrows). Shown here is a silver-stained polyacrylamide gel containing fractions from the purification. Samples for the first seven lanes are listed as follows: mw, molecular weight markers; AS supe, 40% AS supernatant (5 μg protein; 4 U of specific activity); PS pool, phenyl sepharose pool of active fractions (4 μg; 10 U); hep pool, heparin pool (4.8 μg; 300 U); monoS 1, first monoS column (5 μg; 600 U); sup6, gel filtration/superose 6 (2.5 μg; 600 U). The remaining lanes (monoS2) represent fractions from the second monoS column; depolymerizing activity peaked in fractions 16, 17, and 18 (see B). Lanes loaded with these fractions each contain ∼1.3 μg of total protein and 1,260 U of specific activity. Arrows indicate p130 and p160 bands. Identity of the p130 bands (lower arrows) from these fractions was determined by mass spectrometry. (B) Activity profile for fractions for the monoS2 step. Relative activity was estimated for fractions 8–24 by serial titration in the depolymerization assay and is presented in this graph as arbitrary units per microgram protein. (C) p160 and p130 bands are NH2-terminal fragments of XMAP215. Western blots of high-speed supernatant (hss) and monoS2 fractions were probed for XMAP215 with NH2-terminal– or COOH-terminal–specific antibodies.
Mentions: The CPP MT–depolymerizing factor was purified using seven steps: AS precipitation, phenyl sepharose, heparin, monoS, gel filtration, monoQ (pH 7.2), and a final monoS column. When fractions were separated by SDS-PAGE and silver stained, a set of polypeptides of ∼130 kD and a protein of ∼160 kD consistently coeluted with activity on the last two columns in the purification (Fig. 2, A and B , arrows). We estimated that specific activity was enriched several thousand fold by the final monoS step (Table I).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Harvard Medical School, 250 Longwood Ave., Boston, MA 02115, USA. mshirasu@hms.harvard.edu

ABSTRACT
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.

Show MeSH