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Light chain-dependent regulation of Kinesin's interaction with microtubules.

Verhey KJ, Lizotte DL, Abramson T, Barenboim L, Schnapp BJ, Rapoport TA - J. Cell Biol. (1998)

Bottom Line: A pH shift from 7.2 to 6.8 releases inhibition of kinesin without changing its sedimentation behavior.Endogenous kinesin in COS cells also shows pH-sensitive inhibition of MT binding.Taken together, our results provide evidence that a function of LC is to keep kinesin in an inactive ground state by inducing an interaction between the tail and motor domains of HC; activation for cargo transport may be triggered by a small conformational change that releases the inhibition of the motor domain for MT binding.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
We have investigated the mechanism by which conventional kinesin is prevented from binding to microtubules (MTs) when not transporting cargo. Kinesin heavy chain (HC) was expressed in COS cells either alone or with kinesin light chain (LC). Immunofluorescence microscopy and MT cosedimentation experiments demonstrate that the binding of HC to MTs is inhibited by coexpression of LC. Association between the chains involves the LC NH2-terminal domain, including the heptad repeats, and requires a region of HC that includes the conserved region of the stalk domain and the NH2 terminus of the tail domain. Inhibition of MT binding requires in addition the COOH-terminal 64 amino acids of HC. Interaction between the tail and the motor domains of HC is supported by sedimentation experiments that indicate that kinesin is in a folded conformation. A pH shift from 7.2 to 6.8 releases inhibition of kinesin without changing its sedimentation behavior. Endogenous kinesin in COS cells also shows pH-sensitive inhibition of MT binding. Taken together, our results provide evidence that a function of LC is to keep kinesin in an inactive ground state by inducing an interaction between the tail and motor domains of HC; activation for cargo transport may be triggered by a small conformational change that releases the inhibition of the motor domain for MT binding.

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Functional domains of kinesin and models for the activation of kinesin in vivo. (A) Representation of the domains of  kinesin HC and LC, drawn to scale, and the functions assigned to  them. (B) Possible mechanisms to explain the activation of kinesin for MT binding in vivo. Only one HC and one LC of the kinesin tetramer are shown for simplicity. Soluble kinesin in the cell is  in an inactive folded conformation such that the HC tail domain  inhibits MT binding of the motor domain. Activation of kinesin,  represented here by a hypothetical opening of the folded molecule, may be triggered: (A) by interaction of the motor with its  cargo, (B) before or after (as drawn) cargo binding by posttranslational modification (e.g., phosphorylation) of either the HC or  LC, or (C) after cargo binding by a localized shift of pH around  the vesicle from neutral to slightly acidic.
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Figure 9: Functional domains of kinesin and models for the activation of kinesin in vivo. (A) Representation of the domains of kinesin HC and LC, drawn to scale, and the functions assigned to them. (B) Possible mechanisms to explain the activation of kinesin for MT binding in vivo. Only one HC and one LC of the kinesin tetramer are shown for simplicity. Soluble kinesin in the cell is in an inactive folded conformation such that the HC tail domain inhibits MT binding of the motor domain. Activation of kinesin, represented here by a hypothetical opening of the folded molecule, may be triggered: (A) by interaction of the motor with its cargo, (B) before or after (as drawn) cargo binding by posttranslational modification (e.g., phosphorylation) of either the HC or LC, or (C) after cargo binding by a localized shift of pH around the vesicle from neutral to slightly acidic.

Mentions: With the exception of the TPR motifs of LC, all notable domains in both HC and LC can now be assigned a function in either motor activity or in the interaction between the two chains (Fig. 9 A). It therefore seems likely that the TPRs are involved in the one crucial motor function that thus far lacks a corresponding domain; namely, cargo binding. We consider it less likely that the COOH-terminal 10 amino acids of LC, which differ among the various splice forms, are responsible for cargo selection. The proposed role for the TPRs would be consistent with their high conservation across species (Gindhart and Goldstein, 1996), and the fact that TPRs are involved in protein–protein interactions (Lamb et al., 1995; Das et al., 1998). In support of this proposal, an antibody to LC inhibits the binding of purified chick brain kinesin to membranes in vitro (Yu et al., 1992) and an antibody to the third TPR of rat LC releases kinesin from a microsomal vesicle fraction (Stenoien and Brady, 1997). However, other studies have implicated the stalk and tail domains of HC in membrane binding. Purified sea urchin kinesin HC and a bacterially expressed HC stalk-tail domain, but not the stalk domain alone, were able to bind to membranes in vitro (Skoufias et al., 1994). Clearly, a definitive identification of the cargo binding domain has yet to be achieved and it is possible that both the TPR domain of LC and the tail domain of HC are involved in the interaction.


Light chain-dependent regulation of Kinesin's interaction with microtubules.

Verhey KJ, Lizotte DL, Abramson T, Barenboim L, Schnapp BJ, Rapoport TA - J. Cell Biol. (1998)

Functional domains of kinesin and models for the activation of kinesin in vivo. (A) Representation of the domains of  kinesin HC and LC, drawn to scale, and the functions assigned to  them. (B) Possible mechanisms to explain the activation of kinesin for MT binding in vivo. Only one HC and one LC of the kinesin tetramer are shown for simplicity. Soluble kinesin in the cell is  in an inactive folded conformation such that the HC tail domain  inhibits MT binding of the motor domain. Activation of kinesin,  represented here by a hypothetical opening of the folded molecule, may be triggered: (A) by interaction of the motor with its  cargo, (B) before or after (as drawn) cargo binding by posttranslational modification (e.g., phosphorylation) of either the HC or  LC, or (C) after cargo binding by a localized shift of pH around  the vesicle from neutral to slightly acidic.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132950&req=5

Figure 9: Functional domains of kinesin and models for the activation of kinesin in vivo. (A) Representation of the domains of kinesin HC and LC, drawn to scale, and the functions assigned to them. (B) Possible mechanisms to explain the activation of kinesin for MT binding in vivo. Only one HC and one LC of the kinesin tetramer are shown for simplicity. Soluble kinesin in the cell is in an inactive folded conformation such that the HC tail domain inhibits MT binding of the motor domain. Activation of kinesin, represented here by a hypothetical opening of the folded molecule, may be triggered: (A) by interaction of the motor with its cargo, (B) before or after (as drawn) cargo binding by posttranslational modification (e.g., phosphorylation) of either the HC or LC, or (C) after cargo binding by a localized shift of pH around the vesicle from neutral to slightly acidic.
Mentions: With the exception of the TPR motifs of LC, all notable domains in both HC and LC can now be assigned a function in either motor activity or in the interaction between the two chains (Fig. 9 A). It therefore seems likely that the TPRs are involved in the one crucial motor function that thus far lacks a corresponding domain; namely, cargo binding. We consider it less likely that the COOH-terminal 10 amino acids of LC, which differ among the various splice forms, are responsible for cargo selection. The proposed role for the TPRs would be consistent with their high conservation across species (Gindhart and Goldstein, 1996), and the fact that TPRs are involved in protein–protein interactions (Lamb et al., 1995; Das et al., 1998). In support of this proposal, an antibody to LC inhibits the binding of purified chick brain kinesin to membranes in vitro (Yu et al., 1992) and an antibody to the third TPR of rat LC releases kinesin from a microsomal vesicle fraction (Stenoien and Brady, 1997). However, other studies have implicated the stalk and tail domains of HC in membrane binding. Purified sea urchin kinesin HC and a bacterially expressed HC stalk-tail domain, but not the stalk domain alone, were able to bind to membranes in vitro (Skoufias et al., 1994). Clearly, a definitive identification of the cargo binding domain has yet to be achieved and it is possible that both the TPR domain of LC and the tail domain of HC are involved in the interaction.

Bottom Line: A pH shift from 7.2 to 6.8 releases inhibition of kinesin without changing its sedimentation behavior.Endogenous kinesin in COS cells also shows pH-sensitive inhibition of MT binding.Taken together, our results provide evidence that a function of LC is to keep kinesin in an inactive ground state by inducing an interaction between the tail and motor domains of HC; activation for cargo transport may be triggered by a small conformational change that releases the inhibition of the motor domain for MT binding.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
We have investigated the mechanism by which conventional kinesin is prevented from binding to microtubules (MTs) when not transporting cargo. Kinesin heavy chain (HC) was expressed in COS cells either alone or with kinesin light chain (LC). Immunofluorescence microscopy and MT cosedimentation experiments demonstrate that the binding of HC to MTs is inhibited by coexpression of LC. Association between the chains involves the LC NH2-terminal domain, including the heptad repeats, and requires a region of HC that includes the conserved region of the stalk domain and the NH2 terminus of the tail domain. Inhibition of MT binding requires in addition the COOH-terminal 64 amino acids of HC. Interaction between the tail and the motor domains of HC is supported by sedimentation experiments that indicate that kinesin is in a folded conformation. A pH shift from 7.2 to 6.8 releases inhibition of kinesin without changing its sedimentation behavior. Endogenous kinesin in COS cells also shows pH-sensitive inhibition of MT binding. Taken together, our results provide evidence that a function of LC is to keep kinesin in an inactive ground state by inducing an interaction between the tail and motor domains of HC; activation for cargo transport may be triggered by a small conformational change that releases the inhibition of the motor domain for MT binding.

Show MeSH
Related in: MedlinePlus