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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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MT cosedimentation of HC expressed with the LC deletion mutants. Taxol-stabilized MTs and AMP-PNP were added  to lysates from COS cells expressing HC alone (H), HC and LC  (H+L), or HC and the LC deletion mutants (H+L176, H+L237,  H+L405, and H+L488). MTs and bound proteins were sedimented through a sucrose cushion. The MT pellets (P) and supernatants (S) were immunoblotted to detect the expressed proteins  using polyclonal antibodies to the myc- and HA-tags.
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Figure 5: MT cosedimentation of HC expressed with the LC deletion mutants. Taxol-stabilized MTs and AMP-PNP were added to lysates from COS cells expressing HC alone (H), HC and LC (H+L), or HC and the LC deletion mutants (H+L176, H+L237, H+L405, and H+L488). MTs and bound proteins were sedimented through a sucrose cushion. The MT pellets (P) and supernatants (S) were immunoblotted to detect the expressed proteins using polyclonal antibodies to the myc- and HA-tags.

Mentions: MT cosedimentation assays confirmed that the NH2-terminal domain of LC is required for inhibition of MT binding. Both intact LC and the TPR deletion constructs prevented HC from binding to MTs in the presence of AMP-PNP (Fig. 5). The expressed proteins did not pellet with MTs in the presence of ATP (data not shown). Since LC lacking the heptad repeats (LΔHR) is insoluble even in the presence of HC, only HC was detected in the supernatant after centrifugation and, as expected, bound to MTs in an ATP-sensitive manner (data not shown). Taken together, these results indicate that the NH2-terminal region of LC, including the heptad repeats, is required for both the interaction with HC and inhibition of the binding of HC to MTs.


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)

MT cosedimentation of HC expressed with the LC deletion mutants. Taxol-stabilized MTs and AMP-PNP were added  to lysates from COS cells expressing HC alone (H), HC and LC  (H+L), or HC and the LC deletion mutants (H+L176, H+L237,  H+L405, and H+L488). MTs and bound proteins were sedimented through a sucrose cushion. The MT pellets (P) and supernatants (S) were immunoblotted to detect the expressed proteins  using polyclonal antibodies to the myc- and HA-tags.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: MT cosedimentation of HC expressed with the LC deletion mutants. Taxol-stabilized MTs and AMP-PNP were added to lysates from COS cells expressing HC alone (H), HC and LC (H+L), or HC and the LC deletion mutants (H+L176, H+L237, H+L405, and H+L488). MTs and bound proteins were sedimented through a sucrose cushion. The MT pellets (P) and supernatants (S) were immunoblotted to detect the expressed proteins using polyclonal antibodies to the myc- and HA-tags.
Mentions: MT cosedimentation assays confirmed that the NH2-terminal domain of LC is required for inhibition of MT binding. Both intact LC and the TPR deletion constructs prevented HC from binding to MTs in the presence of AMP-PNP (Fig. 5). The expressed proteins did not pellet with MTs in the presence of ATP (data not shown). Since LC lacking the heptad repeats (LΔHR) is insoluble even in the presence of HC, only HC was detected in the supernatant after centrifugation and, as expected, bound to MTs in an ATP-sensitive manner (data not shown). Taken together, these results indicate that the NH2-terminal region of LC, including the heptad repeats, is required for both the interaction with HC and inhibition of the binding of HC to MTs.

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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