Journal of Molecular Biology
Volume 316, Issue 3, 22 February 2002, Pages 817-828
Journal home page for Journal of Molecular Biology

Regular article
XKCM1 acts on a single protofilament and requires the C terminus of tubulin1

https://doi.org/10.1006/jmbi.2001.5360Get rights and content

Abstract

The stability of microtubules during the cell-cycle is regulated by a number of cellular factors, some of which stabilize microtubules and others that promote breakdown. XKCM1 is a kinesin-like protein that induces microtubule depolymerization and is required for mitotic spindle assembly. We have examined the binding and depolymerization effects of XKCM1 on different tubulin polymers in order to learn about its mechanism of action. Zinc-induced tubulin polymers, characterized by an anti-parallel protofilament arrangement, are depolymerized by XKCM1, indicating that this enzyme acts on a single protofilament. GDP-tubulin rings, which correspond to the low-energy state of tubulin, are stable only under conditions that inhibit XKCM1 depolymerizing activity, but can be stabilized by XKCM1 bound to AMPPNP. Tubulin polymers made of subtilisin-treated tubulin (lacking the tubulin C-terminal tail) are resistant to XKCM1-induced depolymerization, suggesting that the interaction of the acidic tail of tubulin with basic residues in XKCM1 unique to Kin I proteins is required for depolymerization.

Introduction

Microtubules (MTs) are critical components of all eukaryotic cells. They are involved in a wide variety of cellular functions, ranging from segregation of genetic material during mitosis to organelle transport and cell movement. MTs are polymers of αβ-tubulin heterodimers that arrange in a head-to-tail fashion into protofilaments. Physiological MTs are 25 nm in diameter and typically contain 13 protofilaments that associate in a parallel fashion to form the hollow structure of the microtubule. Two characteristics of MTs are pivotal in their cellular function. First, due to the head-to-tail arrangement of tubulin heterodimers in parallel protofilaments, MTs are polar. This property enables motor proteins to use MTs as tracks for transport of cargo in a specific direction. Second, MTs, both in vivo and in vitro, exhibit dynamic instability, a behavior in which MTs coexist in states of growth and shrinkage and interconvert randomly between these two states1. Dynamic instability is driven by GTP hydrolysis on β-tubulin within the MT lattice. Polymerized GDP tubulin has a preferred curved conformation2, 3, but within the microtubule lattice, it is thought to be kept straight by lateral contacts between the protofilaments4. Growing MTs contain a stabilizing cap of GTP-bound subunits that are thought to stabilize the microtubule ends. Upon loss of this GTP cap, protofilaments relax into their curved conformation and peel off5, resulting in rapid microtubule depolymerization.

Given the important role that microtubule dynamic instability plays in cytoskeletal function, this property is regulated extensively by cellular factors during the cell-cycle. In particular, the frequency of microtubule catastrophe (switch from growth to shrinkage) is increased significantly in MTs during mitosis relative to interphase, suggesting the existence of microtubule-destabilizing enzymes in the cell6, 7. One such enzyme is XKCM1, identified in Xenopus eggs as a regulator of microtubule dynamics during mitosis8. Its inhibition in extracts results in a fourfold reduction of the frequency of catastrophe, giving rise to long MTs that disrupt mitotic spindle assembly8.

XKCM1 is a member of the Kin I subfamily of kinesins. Unlike conventional kinesins, which convert the chemical energy of ATP hydrolysis into mechanical force for movement along MTs9, the Kin I kinesins exhibit no motile activity and function solely as microtubule-destabilizing enzymes10, 11. XKCM1 directly targets microtubule ends and causes microtubule depolymerization10. This effect most likely occurs by promoting a destabilizing conformational change in the tubulin subunits. It has been proposed that ATP hydrolysis is not required for the destabilizing effect of XKCM1 on the microtubule, but rather is necessary for dissociation of XKCM1 from tubulin to permit recycling of the enzyme. XKIF2, and possibly other members of the Kin I subfamily, exhibit similar microtubule regulatory activity, suggesting that Kin I kinesins may be a major class of microtubule-destabilizers10, 11, 12. A detailed study of the mechanism of action of such regulators is necessary to further our understanding of regulation of cytoskeletal dynamics. In addition, microtubule dynamic regulators can be potential targets for antimitotic drugs that can be employed in therapeutic strategies.

To understand how XKCM1 functions, we have tested its ability to depolymerize different tubulin polymers (Figure 1) to test models of how it induces microtubule depolymerization. In zinc-induced sheets (Figure 1(b), left) and macrotubes (Figure 1(b), right) the individual protofilament conformation is similar to that in MTs, but the protofilaments are arranged in an anti-parallel fashion 13, 14, 15 with the lumenal and external faces of the protofilaments alternating. This topology has allowed us to discriminate between two possible general models of XKCM1-induced depolymerization; simultaneous binding of XKCM1 to a single or to two adjacent protofilaments. Closed ring polymers (Figure 1(c)), which resemble the depolymerized state of tubulin, are formed by GDP-tubulin in the presence of various divalent cations2, 16, 17. These structures are similar to the observed curls of protofilaments induced by XKCM1 depolymerization of GMPCPP-MTs10, and were exploited to increase our understanding of how XKCM1 binds to tubulin and MTs.

Our results show that XKCM1 is able to depolymerize tubulin zinc macrotubes. These results suggest that XKCM1 binds along a single protofilament during the depolymerization cycle. We find that the C terminus of tubulin is essential for the depolymerization activity of XKCM1, and that XKCM1 in the presence of a non-hydrolyzable ATP analog stabilized GDP-tubulin rings. Based on our results from depolymerization experiments as well as some structural modeling, we propose a model for how XKCM1 can induce depolymerization and what features of XKCM1 and the microtubule are essential for this biochemical activity.

Section snippets

Effect of XKCM1 on zinc-induced tubulin polymers versus MTs

We first tested the depolymerization activity of XKCM1 on MTs using a simple pelleting assay (Figure 2). When unpolymerized tubulin and XKCM1 are incubated together, XKCM1 was found in the soluble fraction along with unpolymerized tubulin (Figure 2, lanes 1 and 2). In the absence of XKCM1, but under conditions that promote microtubule assembly, the tubulin was now found in the pellet (Figure 2, lanes 3 and 4). When XKCM1 was added to a microtubule solution, XKCM1 quickly associated with MTs and

XKCM1 acts on a single protofilament

XKCM1, a member the Kin I subfamily of kinesins, exhibits no motor activity but rather functions as a microtubule-depolymerizing enzyme. The binding of XKCM1 to MTs in vitro is sufficient to induce depolymerization, while ATP hydrolysis is most likely involved in recycling the enzyme from a complex with tubulin10. In our study, we tested two general models for how XKCM1 binding could cause a depolymerizing conformational change in MTs: whether XKCM1 dimers bind two adjacent protofilaments and

Conclusions

The present study strongly supports a model in which XKCM1 binding to tubulin is restricted to a single protofilament. While the enzyme can still bind to microtubules that lack the C-terminal tail of tubulin, this acidic segment is required for the XKCM1 destabilizing action. Conserved regions within the catalytic core unique to Kin I proteins that are rich in basic residues are located on the surface facing the microtubule and in positions where they are likely to interact with the acidic

Tubulin and XKCM1 purification

Porcine brain tubulin was purified by several warm/cold cycling steps as described34. Aliquots of tubulin (18 mg/ml) in tubulin buffer I (TB I: 80 mM Pipes, 1 mM EGTA, 1 mM MgCl2) were frozen and stored at −80°C. MAP-free bovine tubulin in TB I at 10 mg/ml from Cytoskeleton, Inc. (Boulder, CO) was used in some cases. XKCM1 was purified from an Sf-9/baculovirus expression system through a combination of ion-exchange and gel-filtration chromatography as described10. XKCM1 fractions eluted from

Acknowledgements

We thank Jan Paluh and Stephanie Ems-McClung for their valuable comments on the manuscript. This work was supported by grants from the NIH and American Heart Association- Midwest Affiliate (to C.E.W.) and by the Office of Health and Enviromental Research of the U.S. Department of Energy (to E.N.). C.E.W. is a Scholar of the Leukemia and Lymphoma Society.

References (37)

  • M.R. Mejillano et al.

    Assembly properties of tubulin after carboxyl group modification

    J. Biol. Chem.

    (1991)
  • E. Mandelkow et al.

    Structures of kinesin and kinesin-microtubule interactions

    Curr. Opin. Cell Biol.

    (1999)
  • S. Lobert et al.

    Subtilisin cleavage of tubulin heterodimers and polymers

    Arch. Biochem. Biophys.

    (1992)
  • V. Peyrot et al.

    C-terminal cleavage of tubulin by subtilisin enhances ring formation

    Arch. Biochem. Biophys.

    (1990)
  • T. Mitchison et al.

    Dynamic instability of microtubule growth

    Nature

    (1984)
  • W.D. Howard et al.

    GDP state of tubulinstabilization of double rings

    Biochemistry (Moscow)

    (1986)
  • R. Melki et al.

    Cold depolymerization of microtubules to double ringsgeometric stabilization of assemblies

    Biochemistry (Moscow)

    (1989)
  • M. Caplow et al.

    The free energy of hydrolysis of a microtubule-bound nucleotide triphosphate is near zeroall of the free energy for hydrolysis is stored in the microtubule lattice

    J. Cell Biol.

    (1994)
  • Cited by (63)

    • Mechanism of Catalytic Microtubule Depolymerization via KIF2-Tubulin Transitional Conformation

      2017, Cell Reports
      Citation Excerpt :

      A large excess of KIF2 (molar ratio KIF2Acore:tubulin dimer = 1:100) resulted in disassembly of tubulin pf-rings in the absence of ATP hydrolysis (Figure 1C, High Conc.). These data indicated that KIF2Acore possesses a fundamental function of binding the curled tubulin protofilament without the completion of ATP hydrolysis; however, a small amount of KIF2 requires the completion of ATP hydrolysis to execute catalytic depolymerization, a finding that is consistent with those from previous reports (Desai et al., 1999; Friel and Howard, 2011; Helenius et al., 2006; Moores and Milligan, 2008; Moores et al., 2002; Niederstrasser et al., 2002; Wagenbach et al., 2008). Furthermore, using AFM, we monitored the moment of catalytic depolymerization of the pf-ring by KIF2Acore in the presence of ATP (KIF2Acore:tubulin = 1:2,000).

    • A Hypothesis on the Origin and Evolution of Tubulin

      2013, International Review of Cell and Molecular Biology
    • Kinesin structure and biochemistry

      2012, Comprehensive Biophysics
    • Regulation of microtubule dynamics by kinesins

      2011, Seminars in Cell and Developmental Biology
      Citation Excerpt :

      Kinesins-13 are not active translocases [14]. They accumulate at microtubule ends either by diffusion along microtubules [15,16], by direct binding [17] or transport by a translocase kinesin [18]; (Fig. 1) causing a curvature of the tubulin heterodimers, which promotes heterodimer dissociation from the microtubule [14] [19–21]. Depolymerisation of stabilised microtubules may mimic removal of the GTP cap in dynamic microtubules since MCAK also promotes catastrophes in dynamic microtubules [22].

    • Parvulin 17 promotes microtubule assembly by its peptidyl-prolyl cis/trans isomerase activity

      2011, Journal of Molecular Biology
      Citation Excerpt :

      One can speculate that binding to the amphiphilic α-helix may affect the catalytic efficiency of parvulin by determining the tubulin orientation and by increasing the local substrate concentrations. The findings that the amphiphilic α-helix interacts with negatively charged surfaces provide also a first hint for the involvement of the tubulin C-terminus in the tubulin–parvulin interaction because the C-terminus of tubulin is composed of an acidic, solvent-exposed flexible region spanning 10 amino acid residues on α-tubulin and 18 residues on β-tubulin.43–45 Due to its high content of acidic residues, it could compete with DNA and heparin for parvulin binding at this α-helix.

    View all citing articles on Scopus
    1

    Edited by J. Karn

    These authors contributed equally to this work.

    View full text